New Hypothesis Insight of Structural Properties on Four Tau Peptide Fragments in Alzheimer's Disease: Origin from the Histidine Behaviors.

During protein folding and misfolding, structural properties and aggregation tendency can be significantly influenced by histidine behaviors (tautomeric behaviors and protonation behaviors). The original reasons were derived from the net charge changes and the various N/N-H orientation on imidazole rings. In the current study, total 18 independent REMD simulations were performed to investigate the histidine behaviors on four Tau peptide fragments (MBD, including R1, R2, R3, and R4 fragments). We found that, compared to R1, R2, R3 except (ϵδ), and R4 systems with flexible structural features, only R3(ϵδ) has dominating conformational structure (possibility of 81.3 %) with three ß-strand structures in parallel ß-sheet structures at I4-K6 and I24-H26, as well as antiparallel ß-sheet structure at G19-L21. Importantly, the H25 and H26 residues (in R3(ϵδ) system) are directly involved in the sheet structure formations and strong H-bonded interactions (possibility range of 31.3 %-44.7 %). Furthermore, the donors and acceptors analysis confirmed that only R3(ϵδ) shows faraway amino acids interaction features in both H25 and H26 residues, and such cooperation effects of two histidine residues contribute to current structural features. The current study will be helpful to further enrichment of the histidine behavior hypothesis, it provides new insight for understanding protein folding and misfolding.

Concentration Effect, Structural Properties, and Driving Force on Aß28 Dimerization with and without Zn2+ Cooperation: Learning from Replica Exchange Sampling.

Zn2+ is a very important factor in promoting the formation of amyloid beta (Aß) aggregates and amyloid plaques. The Zn2+ -bound Aß species generate amorphous or low molecular-weight oligomers. However, it is a lack of studies to approach the starting structural features (dimerization) in Aß nucleation processes with and without Zn2+ , which is the key point in understanding Zn2+ -induced nucleation mechanisms. To better understand the effect of concentration, structural properties, and the driving force, 14 independent replica exchange molecular dynamics simulations were performed in Aß28 dimerization with and without Zn2+ (zAß28 ) cooperation. Our scanning results show that the aggregation propensity is easier in Aß28 -Aß28 and Aß28 -zAß28 systems than zAß28 -zAß28 system. In binding property, the Aß28 -Aß28 model (-61.5 kcal mol-1 ) is stronger than zAß28 -zAß28 (-26.6 kcal mol-1 ) and Aß28 -zAß28 (-7.24 kcal mol-1 ) models. Further analysis confirmed that H13 and H14 residues play specific roles in the three systems. The key point is the orientation of N atom of the imidazole ring in histidine residues. Furthermore, we discovered different driving forces for each system. Our current study contributes to the understanding of how the Aß28 dimer interacts with Zn2+ , which could lead to new insights into Zn2+ -induced nucleation mechanisms.

Ultrastructural evidence for self-replication of Alzheimer-associated Aß42 amyloid along the sides of fibrils.

The nucleation of Alzheimer-associated Aß peptide monomers can be catalyzed by preexisting Aß fibrils. This leads to autocatalytic amplification of aggregate mass and underlies self-replication and generation of toxic oligomers associated with several neurodegenerative diseases. However, the nature of the interactions between the monomeric species and the fibrils during this key process, and indeed the ultrastructural localization of the interaction sites have remained elusive. Here we used NMR and optical spectroscopy to identify conditions that enable the capture of transient species during the aggregation and secondary nucleation of the Aß42 peptide. Cryo-electron microscopy (cryo-EM) images show that new aggregates protrude from the entire length of the progenitor fibril. These protrusions are morphologically distinct from the well-ordered fibrils dominating at the end of the aggregation process. The data provide direct evidence that self-replication through secondary nucleation occurs along the sides of fibrils, which become heavily decorated under the current solution conditions (14 µM Aß42, 20 mM sodium phosphate, 200 µM EDTA, pH 6.8).

Identification of Aggregation Processes in Hexamethylenetetramine Aqueous Solutions: A Comprehensive Raman and Acoustic Spectroscopic Study Combined with Density Functional Theory Calculations.

Raman scattering has been employed to study in detail the concentration dependence of the vibrational modes for hexamethylenetetramine (HMTA) aqueous solutions. The formation of protonated and/or aggregated species has been clarified by comparing the experimental with the theoretically predicted vibrational spectra by means of quantum mechanical calculations. The analysis has shown that the vibrational modes of the solutions arise from a contribution of the vibrational modes of the HMTA self-aggregates and hetero-aggregates of HMTA with water molecules that are formed in the low- and intermediate-concentration regions, respectively. The protonation of HMTA is ruled out due to the large differences between the experimental and the theoretically calculated spectra of the protonated molecules of HTMA in the fingerprint region. In the low-concentration solutions, the hetero-aggregation reaction of HMTA with water is the dominant mechanism, while at higher concentrations, a self-aggregation mechanism occurs. Ultrasonic absorption and velocity measurements were carried out for hexamethylenetetramine aqueous solutions. The acoustic spectra reveal the presence of only one single Debye-type relaxation process that is assigned to the aggregation mechanism of HMTA. The sound absorption data follow two different dependencies on the HMTA mole fraction. The crossover 0.018 mole fraction signifies two separate regions with distinct structural characteristics. The relaxation mechanism observed in dilute solutions was attributed to hetero-association of HMTA with water molecules, while at higher concentrations, the observed relaxation process was assigned to the self-association reaction of HMTA molecules. This structural transformation is also reflected in several physicochemical properties of the system, including the kinematic viscosity, the mass density, the sound speed and the adiabatic compressibility of the HMTA aqueous solutions. The combination of vibrational and acoustic spectroscopies with molecular orbital calculations allowed us to disentangle the underlying processes and to elucidate the observed relaxation mechanism in the HMTA aqueous solutions.

Hierarchical Attention Neural Network for Event Types to Improve Event Detection.

Event detection is an important task in the field of natural language processing, which aims to detect trigger words in a sentence and classify them into specific event types. Event detection tasks suffer from data sparsity and event instances imbalance problems in small-scale datasets. For this reason, the correlation information of event types can be used to alleviate the above problems. In this paper, we design a Hierarchical Attention Neural Network for Event Types (HANN-ET). Specifically, we select Long Short-Term Memory (LSTM) as the semantic encoder and utilize dynamic multi-pooling and the Graph Attention Network (GAT) to enrich the sentence feature. Meanwhile, we build several upper-level event type modules and employ a weighted attention aggregation mechanism to integrate these modules to obtain the correlation event type information. Each upper-level module is completed by a Neural Module Network (NMNs), event types within the same upper-level module can share information, and an attention aggregation mechanism can provide effective bias scores for the trigger word classifier. We conduct extensive experiments on the ACE2005 and the MAVEN datasets, and the results show that our approach outperforms previous state-of-the-art methods and achieves the competitive F1 scores of 78.9% on the ACE2005 dataset and 68.8% on the MAVEN dataset.

Dispersal network heterogeneity promotes species coexistence in hierarchical competitive communities.

Understanding the mechanisms of biodiversity maintenance is a fundamental issue in ecology. The possibility that species disperse within the landscape along differing paths presents a relatively unexplored mechanism by which diversity could emerge. By embedding a classical metapopulation model within a network framework, we explore how access to different dispersal networks can promote species coexistence. While it is clear that species with the same demography cannot coexist stably on shared dispersal networks, we find that coexistence is possible on unshared networks, as species can surprisingly form self-organised clusters of occupied patches with the most connected patches at the core. Furthermore, a unimodal biodiversity response to an increase in species colonisation rates or average patch connectivity emerges in unshared networks. Increasing network size also increases species richness monotonically, producing characteristic species-area curves. This suggests that, in contrast to previous predictions, many more species can co-occur than the number of limiting resources.

On the role of sidechain size and charge in the aggregation of Aß42 with familial mutations.

The aggregation of the amyloid-ß (Aß) peptide is linked to the pathogenesis of Alzheimer's disease (AD). In particular, some point mutations within Aß are associated with early-onset familial Alzheimer's disease. Here we set out to explore how the physical properties of the altered side chains, including their sizes and charges, affect the molecular mechanisms of aggregation. We focus on Aß42 with familial mutations-A21G (Flemish), E22K (Italian), E22G (Arctic), E22Q (Dutch), and D23N (Iowa)-which lead to similar or identical pathology with sporadic AD or severe cerebral amyloid angiopathy. Through global kinetic analysis, we find that for the E22K, E22G, E22Q, and D23N mutations, the acceleration of the overall aggregation originates primarily from the modulation of the nucleation processes, in particular secondary nucleation on the surface of existing fibrils, whereas the elongation process is not significantly affected. Remarkably, the D23 position appears to be responsible for most of the charge effects during nucleation, while the size of the side chain at the E22 position plays a more significant role than its charge. Thus, we have developed a kinetic approach to determine the nature and the magnitude of the contribution of specific residues to the rate of individual steps of the aggregation reaction, through targeted mutations and variations in ionic strength. This strategy can help rationalize the effect of some disease-related mutations as well as yield insights into the mechanism of aggregation and the transition states of the wild-type protein.

Glomalin-related soil protein: The particle aggregation mechanism and its insight into coastal environment improvement.

Glomalin-related soil protein (GRSP), a ubiquitous microbial product, plays a crucial role in particle aggregation and metal adsorption, but the underlying mechanisms remain unknown. Here, GRSP fraction was extracted from estuarine ecosystems and systematically characterized to elucidate the aggregation mechanisms and its impact on coastal environment improvement. We found that GRSP fraction (gravimetric mass of extracted GRSP, 5.1-24.3 mg g-1) was a globally relevant novel bioflocculant and that protein (linked to Bradford protein assay, 1.64-4.37 mg g-1) was the active flocculant constituent. The zeta potential, FTIR, XPS, and 13C NMR analyses identified its key constituents and structure, and revealed that the charge neutralization and bridging were GRSP fraction aggregation mechanisms. Thermogravimetric-infrared spectrometry analysis showed that GRSP fraction was highly thermostable, and the main volatile pyrolysis products included H2O, CO2, CO, and CH4. The SEM-EDX and XPS Fe valence spectroscopy suggested that GRSP fraction contained rich Fe (11.91 ± 0.48%) and could form Fe-rich flocs with particles. We also found that GRSP fraction has a high adsorption capacity (76-95%) for Cu, Zn, Pb and Cd, and its flocculation properties provide new insights into metal adsorption. The analysis of particle aggregation mechanism and its metal adsorption capacity is of great significance to elucidate the role of GRSP fraction in coastal environment improvement.

Lysozyme Fibrils Alter the Mechanism of Insulin Amyloid Aggregation.

Protein aggregation into amyloid fibrils is linked to multiple disorders. The understanding of how natively non-harmful proteins convert to these highly cytotoxic amyloid aggregates is still not sufficient, with new ideas and hypotheses being presented each year. Recently it has been shown that more than one type of protein aggregates may co-exist in the affected tissue of patients suffering from amyloid-related disorders, sparking the idea that amyloid aggregates formed by one protein may induce another protein's fibrillization. In this work, we examine the effect that lysozyme fibrils have on insulin amyloid aggregation. We show that not only do lysozyme fibrils affect insulin nucleation, but they also alter the mechanism of its aggregation.

Differences in nucleation behavior underlie the contrasting aggregation kinetics of the Aß40 and Aß42 peptides.

The two major forms of the amyloid-beta (Aß) peptide found in plaques in patients suffering from Alzheimer's disease, Aß40 and Aß42, only differ by two amino acids in the C-terminal region, yet they display markedly different aggregation behavior. The origins of these differences have remained challenging to connect to specific molecular-level processes underlying the aggregation reaction. In this paper we use a general strategy to apply the conventional workflow of chemical kinetics to the aggregation of the Aß40 peptide to identify the differences between Aß40 and Aß42 in terms of the microscopic determinants of the aggregation reaction. Our results reveal that the major source of aggregates in the case of Aß40 is a fibril-catalyzed nucleation process, the multistep nature of which is evident through its saturation behavior. Moreover, our results show that the significant differences in the observed behavior of the two proteins originate not simply from a uniform increase in all microscopic rates for Aß42 compared with Aß40, but rather are due to a shift of more than one order of magnitude in the relative importance of primary nucleation versus fibril-catalyzed secondary nucleation processes. This analysis sheds light on the microscopic determinants of the aggregation behavior of the principal forms of Aß and outlines a general approach toward achieving an understanding at the molecular level of the aberrant deposition of insoluble peptides in neurodegenerative disorders.

Thermal aggregation behaviour of soy protein: characteristics of different polypeptides and sub-units.

BACKGROUND: Due to the differences in structure and composition of glycinin and ß-conglycinin, they exhibit different characteristics during heat treatment. In present study, the thermal aggregation behaviour of glycinin, ß-conglycinin and their isolated sub-units was investigated at pH 7.0. RESULTS: Acidic polypeptides, basic polypeptides, αα' and ß sub-units of soy protein were denatured during the isolation process. The degree of aggregation of protein fractions after heat treatment was in the order: denatured basic polypeptides > native glycinin > denatured ß sub-unit > native ß-conglycinin > denatured acidic polypeptides > denatured αα' sub-units. Glycinin, ß-conglycinin, acidic polypeptides and αα'/ß sub-units exhibited different changing trends of surface hydrophobicity with increasing temperature. The αα' sub-units showed higher ability to suppress thermal aggregation of basic polypeptides than ß sub-units during heat treatment. The ß sub-units were shown to form soluble aggregates with glycinin after heating. CONCLUSION: The interaction mechanism of αα' and ß sub-units heated with basic polypeptides was proposed. For the ß sub-units-basic polypeptides mixed system, more hydrophobic chains were binding together and buried inside during heat treatment, which resulted in lower surface hydrophobicity. The αα' sub-units-basic polypeptides mixed system was considered to be a stable system with higher surface hydrophobicity after being heated.

Aß42 and Aß40: similarities and differences.

The abnormal accumulation of amyloid-ß (Aß) peptide in the brain is one of the most important hallmarks of Alzheimer's disease. Aß is an aggregation-prone and toxic polypeptide with 39-43 residues, derived from the amyloid precursor protein proteolysis process. According to the amyloid hypothesis, abnormal accumulation of Aß in the brain is the primary influence driving Alzheimer's disease pathologies. Among all kinds of Aß isoforms, Aß40 and Aß42 are believed to be the most important ones. Although these two kinds of Aß differ only in two amino acid residues, recent studies show that they differ significantly in their metabolism, physiological functions, toxicities, and aggregation mechanism. In this review, we mainly summarize the similarities and differences between Aß42 and Aß40, recent studies on selective inhibitors as well as probes will also be mentioned.

Edge Substitution Effects of Histidine Tautomerization Behaviors on the Structural Properties and Aggregation Properties of Aß(1-42) Mature Fibril.

Histidine behaviors play critical roles in folding and misfolding processes due to the changes in net charge and the various N/N-H orientations on imidazole rings. However, the effect of histidine tautomerization (HIE (Nε-H, ε) and HID (Nδ-H, δ) states) behaviors on the edge chain of Aß mature fibrils remains inadequately understood, which is critical for finding a strategy to disturb fibril elongation and growth. In the current study, eight independent molecular dynamics simulations were conducted to investigate such impacts on the structural and aggregation properties. Our results from three different binding models revealed that the binding contributions of edge substitution effects are primarily located between chains 1 and 2. Histidine states significantly influence the secondary structure of each domain. Further analysis confirmed that the C1_H6//C1_E11 intrachain interaction is essential in maintaining the internal stability of chain 1, while the C1_H13//C2_H13 and C1_H14//C2_H13 interchain interactions are critical in maintaining the interchain stability of the fibril structure. Our subsequent analysis revealed that the current edge substitution leads to the loss of the C1_H13//C1_E11 intrachain and C1_H13//C2_H14 interchain interactions. The N-terminal regularity was significantly directly influenced by histidine states, particularly by the residue of C1_H13. Our study provides valuable insights into the effect of histidine behaviors on the edge chain of Aß mature fibril, advancing our understanding of the histidine behavior hypothesis in misfolding diseases.

Multi-scale modeling of natural organic matter-heavy metal cations interactions: Aggregation and stabilization mechanisms.

Interaction between natural organic matters (NOM) and heavy metal cations in aqueous environment are of great significance for maintaining stability of organic carbon and restraining transport of heavy metal contaminants in (bio)geochemical processes. We systematically explore the aggregation process and complexation between NOM and heavy metal cations (Ag+, Cd2+, Pb2+, Zn2+, Eu3+) under different pH condition by molecular dynamics (MD) simulations, umbrella sampling method, and quantum chemistry calculations. The character of molecular structures NOM-heavy metal complexes and association are quantified. In acidic pH condition, aggregation proceeds via H-bonding and π-π interactions between NOM fragments. In neutral condition, Ag+, Cd2+, Pb2+, and Eu3+ can form inner-sphere complexes with the surface carboxylic groups and therefore reduce intermolecular charge repulsion, eventually leading to NOM aggregation, and it shows that even without direct binding, the outer-sphere adsorbed Zn2+ can also result in the formation of NOM assemble through H-bonding. Consequently, these heavy metals are capable of promoting NOM aggregation regardless of the complexing ways. Complexing free energy calculations characterized the dynamic processes of cations binding to the carboxylic groups of NOM fragment and the related energy landscape. This study provides quantitative insights for understanding the environmental processes of heavy metals and cycle of C in aquatic ecosystem, and contributes to developing environment-friendly strategies for controlling heavy metal contaminants.

Understanding the mechanism of amylin aggregation: From identifying crucial segments to tracing dominant sequential events to modeling potential aggregation suppressors.

One of the most abundant, prevailing, and life-threatening human diseases that are currently baffling the scientific community is type 2 diabetes (T2D). The self-association of human amylin has been implicated in the pathogenesis of T2D, though with an inconclusive understanding of the mechanism. Hence, we focused on the characterization of the conformational ensembles of all the species that are believed to define the structural polymorphism of the aggregation process - the functional monomeric, the initially self-associated oligomeric, and the structured protofibril - by employing near-equilibrium, non-equilibrium, and equilibrium atomistic simulations on the sporadic, two familial variants (S20G and G33R), and their proline-substituted forms (S20P and G33P). The dynamic near-equilibrium assays hint toward - the abundance of helical conformation in the monomeric state, the retainment of the helicity in the initial self-associated oligomeric phase pointing toward the existence of the helix-helix association mechanism, the difference in preference of specific segments to have definite secondary structural features, the phase-dependent variability in the dominance of specific segments and mutation sites, and the simultaneous presence of generic and unique features among various sequences. Furthermore, the non-equilibrium pulling assays exemplify a generic sequential unzipping mechanism of the protofibrils, however, the sequence-dependent uniqueness comes from the difference in location and magnitude of the control of a specific terminus. Importantly, the equilibrium thermodynamic assays efficiently rank order the potential of aggregability among sequences and consequently suggests the probability of designing effective aggregation suppressors against sporadic and familial amylin variants incorporating proline as the mutation.

New Insights into the Structural and Binding Properties on Aß Mature Fibrils Due to Histidine Protonation Behaviors.

Histidine tautomeric behaviors have been considered origin factors for controlling the structure and aggregation properties of misfolding peptides. Except for tautomeric behaviors, histidine protonation behaviors definitely have the same capacities due to the net charge changes and the various N/N-H orientations on imidazole rings. However, such phenomena are still unknown. In the current study, Aß mature fibrils substituted with various protonation states were performed by molecular dynamics simulations to investigate the structure and binding properties. Our results show that all kinds of protonation states can increase the ΔG1 stability and decrease ΔG2 and ΔG3 stabilities. A significantly higher averaged ß-sheet content was detected in (εεp), (εpp), and (ppp) fibrils in one, two, and three protonation stages, respectively. Impressively, we found that the substituted fibril with specific protonated states can control the N-terminus structural properties. Further analysis confirmed that H6 and H13 are more important than H14 since the H-bond donor and receptor cooperate among C1/C3/C8_H6, C1/C3/C8_H13, and C1/C3/C8_E11. Furthermore, the mechanism of protonation behaviors was discussed. The current study is helpful for understanding the histidine protonation behaviors on one, two, and three protonation stages, which provides new horizons for exploring the origin of protein folding and misfolding.

Influence of denaturants on amyloid ß42 aggregation kinetics.

Amyloid formation is linked to devastating neurodegenerative diseases, motivating detailed studies of the mechanisms of amyloid formation. For Aß, the peptide associated with Alzheimer's disease, the mechanism and rate of aggregation have been established for a range of variants and conditions in vitro and in bodily fluids. A key outstanding question is how the relative stabilities of monomers, fibrils and intermediates affect each step in the fibril formation process. By monitoring the kinetics of aggregation of Aß42, in the presence of urea or guanidinium hydrochloride (GuHCl), we here determine the rates of the underlying microscopic steps and establish the importance of changes in relative stability induced by the presence of denaturant for each individual step. Denaturants shift the equilibrium towards the unfolded state of each species. We find that a non-ionic denaturant, urea, reduces the overall aggregation rate, and that the effect on nucleation is stronger than the effect on elongation. Urea reduces the rate of secondary nucleation by decreasing the coverage of fibril surfaces and the rate of nucleus formation. It also reduces the rate of primary nucleation, increasing its reaction order. The ionic denaturant, GuHCl, accelerates the aggregation at low denaturant concentrations and decelerates the aggregation at high denaturant concentrations. Below approximately 0.25 M GuHCl, the screening of repulsive electrostatic interactions between peptides by the charged denaturant dominates, leading to an increased aggregation rate. At higher GuHCl concentrations, the electrostatic repulsion is completely screened, and the denaturing effect dominates. The results illustrate how the differential effects of denaturants on stability of monomer, oligomer and fibril translate to differential effects on microscopic steps, with the rate of nucleation being most strongly reduced.

Advances in the understanding of protein misfolding and aggregation through molecular dynamics simulation.

Aberrant protein folding known as protein misfolding is counted as one of the striking factors of neurodegenerative diseases. The extensive range of pathologies caused by protein misfolding, aggregation and subsequent accumulation are mainly classified into either gain of function diseases or loss of function diseases. In order to seek for novel strategies for treatment and diagnosis of neurodegenerative diseases, insights into the mechanism of misfolding and aggregation is essential. A comprehensive knowledge on the factors influencing misfolding and aggregation is required as well. An extensive experimental study on protein aggregation is somewhat challenging due to the insoluble and noncrystalline nature of amyloid fibrils. Thus there has been a growing use of computational approaches including Monte Carlo simulation, docking simulation, molecular dynamics simulation in the study of protein misfolding and aggregation. The review presents a discussion on molecular dynamics simulation alone as to how it has emerged as a promising tool in the understanding of protein misfolding and aggregation in general, detailing upon three different aspects considering four misfold prone proteins in particular. It is noticeable that all four proteins considered in this review i.e prion, superoxide dismutase1, huntingtin and amyloid ß are linked to chronic neurodegenerative diseases with debilitating effects. Initially the review elaborates on the factors influencing the misfolding and aggregation. Next, it addresses our current understanding of the amyloid structures and the associated aggregation mechanisms, finally, summarizing the contribution of this computational tool in the search for therapeutic strategies against the respective protein-deposition diseases.

Early aggregation mechanism of Aß16-22 revealed by Markov state models.

Aß16-22 is believed to have critical role in early aggregation of full length amyloids that are associated with the Alzheimer's disease and can aggregate to form amyloid fibrils. However, the early aggregation mechanism is still unsolved. Here, multiple long-term molecular dynamics simulations combining with Markov state model were used to probe the early oligomerization mechanism of Aß16-22 peptides. The identified dimeric form adopted either globular random-coil or extended ß-strand like conformations. The observed dimers of these variants shared many overall conformational characteristics but differed in several aspects at detailed level. In all cases, the most common type of secondary structure was intermolecular antiparallel ß-sheets. The inter-state transitions were very frequent ranges from few to hundred nanoseconds. More strikingly, those states which contain fraction of ß secondary structure and significant amount of extended coiled structures, therefore exposed to the solvent, were majorly participated in aggregation. The assembly of low-energy dimers, in which the peptides form antiparallel ß sheets, occurred by multiple pathways with the formation of an obligatory intermediates. We proposed that these states might facilitate the Aß16-22 aggregation through a significant component of the conformational selection mechanism, because they might increase the aggregates population by promoting the inter-chain hydrophobic and the hydrogen bond contacts. The formation of early stage antiparallel ß sheet structures is critical for oligomerization, and at the same time provided a flat geometry to seed the ordered ß-strand packing of the fibrils. Our findings hint at reorganization of this part of the molecule as a potentially critical step in Aß aggregation and will insight into early oligomerization for large ß amyloids.

Formation mechanisms and assembly patterns of anammox biofilm induced by carrier type: Novel insights based on low-strength wastewater treatment.

The morphological structure, properties, microbial community and function of anammox biofilms induced by large-pore carriers (Bls), small-pore carriers, filament carriers and non-carriers (Bn) in low-strength wastewater were comprehensively studied. The carriers promoted biomass accumulation and agglomeration, with Bls demonstrating the highest biomass proportion of 0.76, the highest specific anammox activity (0.41 kgN/(kgVSS·d)-1) and the largest aggregates. Hydraulic shearing stimulated Bn to secrete most extracellular polymeric substances and capture more inorganic ions for enhanced strength. Metagenomic sequencing showed that the four biofilms shared a common core flora, but differed in cross-metabolism. The proportion of the functional bacterium Candidatus Brocadia was highest in Bls, while the increase in heterotrophic bacteria in Bn supported stronger metabolic capacity. Finally, the proposed anisotropic or isotropic carrier structure was identified as the key to generating "uniform development" and "central development" models. This study is helpful for understanding the anammox aggregation mechanism and carrier optimization.