There is growing recognition regarding the role of intracellular amyloid beta (Aβ) in the Alzheimer's disease process, which has been linked with aberrant signaling and the disruption of protein degradation mechanisms. Most notably, intraneuronal Aβ likely underlies the oxidative stress and mitochondrial dysfunction that have been identified as key elements of disease progression. In this study, we employed fluorescence imaging to explore the ability of a bifunctional small molecule to reduce aggregates of intracellular Aβ and attenuate oxidative stress. Structurally, this small molecule is comprised of a nitroxide spin label linked to an amyloidophilic fluorene and is known as spin-labeled fluorene (SLF). The effect of the SLF on intracellular Aβ accumulation and oxidative stress was measured in MC65 cells, a human neuronal cell line with inducible expression of the amyloid precursor protein and in the N2a neuronal cell line treated with exogenous Aβ. Super-resolution microscopy imaging showed SLF decreases the accumulation of intracellular Aβ. Confocal microscopy imaging of MC65 cells treated with a reactive oxygen species (ROS)-sensitive dye demonstrated SLF significantly reduces the intracellular Aβ-induced ROS signal. In order to determine the contributions of the separate SLF moieties to these protective activities, experiments were also carried out on cells with nitroxides lacking the Aβ targeting domain or fluorene derivatives lacking the nitroxide functionality. The findings support a synergistic effect of SLF in counteracting both the conformational toxicity of both endogenous and exogenous Aβ, its promotion of ROS, and Aβ metabolism. Furthermore, these studies demonstrate an intimate link between ROS production and Aβ oligomer formation.

The oligomer of β-amyloid (Aβ) is considered the main neurotoxin in Alzheimer's disease (AD). Therefore, the inhibition of the formation of Aβ oligomer could be a target for AD therapy. In this study, with the help of the dot blotting assay and transmission electronic microscopy, it was have discovered that 5-hydroxycyclopenicillone, a cyclopentenone recently isolated from a sponge-associated fungus, effectively reduced the formation of Aβ oligomer from Aβ peptide in vitro. Molecular dynamics simulations suggested hydrophobic interactions between 5-hydroxycyclopenicillone and Aβ peptide, which might prevent the conformational transition and oligomerization of Aβ peptide. Moreover, Aβ oligomer pre-incubated with 5-hydroxycyclopenicillone was less toxic when added to neuronal SH-SY5Y cells compared to the normal Aβ oligomer. Although 5-hydroxycyclopenicillone is not bioavailable in the brain in its current form, further modification or encapsulation of this chemical might improve the penetration of 5-hydroxycyclopenicillone into the brain. Based on the current findings and the anti-oxidative stress properties of 5-hydroxycyclopenicillone, it is suggested that 5-hydroxycyclopenicillone may have potential therapeutic efficacy in treating AD.

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer's and Parkinson's diseases, respectively, a process that is intimately linked to the diseases' progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aβ40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.

Amyloid-β peptide (Aβ) is an intrinsically disordered protein (IDP) associated with Alzheimer's disease. The structural flexibility and aggregation propensity of Aβ pose major challenges for elucidating the interaction between Aβ monomers and ligands. All-D-peptides consisting solely of D-enantiomeric amino acid residues are interesting drug candidates that combine high binding specificity with high metabolic stability. Here we characterized the interaction between the 12-residue all-D-peptide D3 and Aβ42 monomers, and how the interaction influences Aβ42 aggregation. We demonstrate for the first time that D3 binds to Aβ42 monomers with submicromolar affinities. These two highly unstructured molecules are able to form complexes with 1:1 and other stoichiometries. Further, D3 at substoichiometric concentrations effectively slows down the β-sheet formation and Aβ42 fibrillation by modulating the nucleation process. The study provides new insights into the molecular mechanism of how D3 affects Aβ assemblies and contributes to our knowledge on the interaction between two IDPs.

Alzheimer's disease (AD) and Parkinson's disease (PD), including dementia with Lewy bodies (DLB), account for the majority of dementia cases worldwide. Interestingly, a significant number of patients have clinical and neuropathological features of both AD and PD, i.e., the presence of amyloid deposits and Lewy bodies in the neocortex. The identification of α-synuclein peptides in amyloid plaques in DLB brain led to the hypothesis that both peptides mutually interact with each other to facilitate neurodegeneration. In this article, we report the influence of Aβ(1-42) and pGlu-Aβ(3-42) on the aggregation of α-synuclein in vitro. The aggregation of human recombinant α-synuclein was investigated using thioflavin-T fluorescence assay. Fibrils were investigated by means of antibody conjugated immunogold followed by transmission electron microscopy (TEM). Our data demonstrate a significantly increased aggregation propensity of α-synuclein in the presence of minor concentrations of Aβ(1-42) and pGlu-Aβ(3-42) for the first time, but without effect on toxicity on mouse primary neurons. The analysis of the composition of the fibrils by TEM combined with immunogold labeling of the peptides revealed an interaction of α-synuclein and Aβ in vitro, leading to an accelerated fibril formation. The analysis of kinetic data suggests that significantly enhanced nucleus formation accounts for this effect. Additionally, co-occurrence of α-synuclein and Aβ and pGlu-Aβ, respectively, under pathological conditions was confirmed in vivo by double immunofluorescent labelings in brains of aged transgenic mice with amyloid pathology. These observations imply a cross-talk of the amyloid peptides α-synuclein and Aβ species in neurodegeneration. Such effects might be responsible for the co-occurrence of Lewy bodies and plaques in many dementia cases.

Atherosclerosis is characterized by the accumulation of oxidized lipids in the artery wall, which triggers an inflammatory response. Oxidized low-density lipoprotein (ox-LDL) presents amyloid-like structural properties, and different amyloid species have recently been recognized in atherosclerotic plaques. Therefore, we studied the uptake of the amyloid imaging agent [18F]Flutemetamol in atherosclerotic plaques. The binding of [18F]Flutemetamol to human carotid artery plaque was studied in vitro. In vivo uptake of the tracer was studied in hypercholesterolemic IGF-II/LDLR-/-ApoB100/100 mice and C57BL/6N controls. Tracer biodistribution was studied in vivo with PET/CT, and ex vivo by gamma counter and digital ex vivo autoradiography. The presence of amyloid, ox-LDL, and macrophages in the plaques was examined by immunohistochemistry. [18F]Flutemetamol showed specific accumulation in human carotid plaque, especially in areas positive for amyloid beta. The aortas of IGF-II/LDLR-/-ApoB100/100 mice showed large thioflavin-S-positive atherosclerotic plaques containing ox-LDL and macrophages. Autoradiography revealed 1.7-fold higher uptake in the plaques than in a lesion-free vessel wall, but no difference in aortic tissue uptake between mouse strains were observed in the in vivo PET/CT. In conclusion, [18F]Flutemetamol binds to amyloid-positive areas in human atherosclerotic plaques. Further studies are warranted to clarify the uptake mechanisms, and the potential of the tracer for in vivo imaging of atherosclerosis in patients.

Aluminium (Al) is clearly neurotoxic and considerable evidence exists that Al may play a role in the aetiology or pathogenesis of Alzheimer's disease (AD). Nevertheless, the link between AD pathology and Al is still open to debate. Therefore, we investigated here the interaction of aluminium ions with two Aβ peptide fragments and their analogues. First, we synthesised by the Fmoc/tBu solid-phase peptide synthesis (SPPS) strategy using an automated peptide synthesiser two new peptides starting from the Aβ(1-16) native peptide fragment. For this purpose, the three histidine residues (H6, H13, and H14) of the Aβ(1-16) peptide were replaced by three alanine and three serine residues to form the modified peptides Aβ(1-16)A36,13,14 and Aβ(1-16)S36,13,14 (primary structures: H-1DAEFRADSGYEVAAQK16-NH2 and H-1DAEFRSDSGYEVSSQK16-NH2). In addition, the Aβ(9-16) peptide fragment (H-9GYEVHHQK16-NH2) and its glycine analogues, namely Aβ(9-16)G110, (H-9GGEVHHQK16-NH2), Aβ(9-16)G213,14 (H-9GYEVGGQK16-NH2), and Aβ(9-16)G310,13,14 (H-9GGEVGGQK16-NH2), were manually synthesised in order to study Al binding to more specific amino acid residues. Both the peptides and the corresponding complexes with aluminium were comparatively investigated by mass spectrometry (MS), circular dichroism spectroscopy (CD), atomic force microscopy (AFM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). Al-peptide molecular ions and Al-fragment ions were unambiguously identified in the MS and MS/MS spectra. AFM images showed dramatic changes in the film morphology of peptides upon Al binding. Our findings from the investigation of N-terminal 1-16 and even 9-16 normal and modified sequences of Aβ peptides suggest that they have the capability to be involved in aluminium ion binding associated with AD.

Conformational diseases represent a new aspect of proteomic medicine where diagnostic and therapeutic paradigms are evolving. In this context, the early biomarkers for target cell failure (neurons, β-cells, etc.) represent a challenge to translational medicine and play a multidimensional role as biomarkers and potential therapeutic targets. This systematic review, which follows the PICO and Prisma methods, analyses this new-fangled multidimensionality, its strengths and limitations, and presents the future possibilities it opens up. The nuclear diagnosis methods are immunoassays: ELISA, immunodot, western blot, etc., while the therapeutic approach is focused on pharmaco- and molecular chaperones.

Human islet amyloid peptide (hIAPP1-37) aggregation is an early step in Diabetes Mellitus. We aimed to evaluate a family of pharmaco-chaperones to act as modulators that provide dynamic interventions and the multi-target capacity (native state, cytotoxic oligomers, protofilaments and fibrils of hIAPP1-37) required to meet the treatment challenges of diabetes. We used a cross-functional approach that combines in silico and in vitro biochemical and biophysical methods to study the hIAPP1-37 aggregation-oligomerization process as to reveal novel potential anti-diabetic drugs. The family of pharmaco-chaperones are modulators of the oligomerization and fibre formation of hIAPP1-37. When they interact with the amino acid in the amyloid-like steric zipper zone, they inhibit and/or delay the aggregation-oligomerization pathway by binding and stabilizing several amyloid structures of hIAPP1-37. Moreover, they can protect cerebellar granule cells (CGC) from the cytotoxicity produced by the hIAPP1-37 oligomers. The modulation of proteostasis by the family of pharmaco-chaperones A-F is a promising potential approach to limit the onset and progression of diabetes and its comorbidities.

Amyloidosis is a relatively rare human disease caused by the deposition of abnormal protein fibres in the extracellular space of various tissues, impairing their normal function. Proteomic analysis of patients' biopsies, developed by Dogan and colleagues at the Mayo Clinic, has become crucial for clinical diagnosis and for identifying the amyloid type. Currently, the proteomic approach is routinely used at National Amyloidosis Centre (NAC, London, UK) and Istituto di Tecnologie Biomediche-Consiglio Nazionale delle Ricerche (ITB-CNR, Milan, Italy). Both centres are members of the European Proteomics Amyloid Network (EPAN), which was established with the aim of sharing and discussing best practice in the application of amyloid proteomics. One of the EPAN's activities was to evaluate the quality and the confidence of the results achieved using different software and algorithms for protein identification. In this paper, we report the comparison of proteomics results obtained by sharing NAC proteomics data with the ITB-CNR centre. Mass spectrometric raw data were analysed using different software platforms including Mascot, Scaffold, Proteome Discoverer, Sequest and bespoke algorithms developed for an accurate and immediate amyloid protein identification. Our study showed a high concordance of the obtained results, suggesting a good accuracy of the different bioinformatics tools used in the respective centres. In conclusion, inter-centre data exchange is a worthwhile approach for testing and validating the performance of software platforms and the accuracy of results, and is particularly important where the proteomics data contribute to a clinical diagnosis.

CRANAD-28, a difluoroboron curcumin analogue, has been demonstrated in earlier reports to successfully label amyloid beta (Aβ) plaques for imaging both ex vivo and in vivo. CRANAD-28's imaging brightness, ability to penetrate the blood brain barrier, and low toxicity make the compound a potentially potent imaging tool in Alzheimer's research. In this study, the Aβ-labeling ability of CRANAD-28 was investigated in further detail using histological staining to assess different criteria, including stained Aβ plaque brightness, Aβ plaque size, and Aβ plaque number count. The results of this study demonstrated CRANAD-28 to be superior across all criteria assessed. Furthermore, CRANAD-28 and IBA-1 antibody were used to label Aβ-plaques and microglia respectively. Statistical analysis with Spearman regression revealed a statistically significant negative correlation between the size of labeled Aβ plaques and surrounding microglia density. This finding provides interesting insight into Aβ plaque and microglia dynamism in AD pathology and corroborates the findings of previous studies. In addition, we found that CRANAD-28 provided distinct spectral signatures for Aβs in the core and periphery of the plaques. Based on the study's results, CRANAD-28 could be considered as an alternative standard for imaging Aβ-plaques in future research studies.

Amyloid precursor protein (APP) at the plasma membrane is internalized via endocytosis and delivered to endo/lysosomes, where neurotoxic amyloid-β (Aβ) is produced via β-, γ-secretases. Hence, endocytosis plays a key role in the processing of APP and subsequent Aβ generation. β-, γ-secretases as well as APP are localized in cholesterol-enriched lipid raft microdomains. However, it is still unclear whether lipid rafts are the site where APP undergoes endocytosis and whether cholesterol levels affect this process. In this study, we found that localization of APP in lipid rafts was increased by elevated cholesterol level. We also showed that increasing or decreasing cholesterol levels increased or decreased APP endocytosis, respectively. When we labeled cell surface APP, APP localized in lipid rafts preferentially underwent endocytosis compared to nonraft-localized APP. In addition, APP endocytosis from lipid rafts was regulated by cholesterol levels. Our results demonstrate for the first time that cholesterol levels regulate the localization of APP in lipid rafts affecting raft-dependent APP endocytosis. Thus, regulating the microdomain localization of APP could offer a new therapeutic strategy for Alzheimer's disease.

Cistanche tubulosa aqueous extract (CTE) is already used as a botanical prescription drug for treating dementia in China. Our previous studies reported that phenylethanoid glycosides of CTE have anti-Alzheimer's disease (AD) activity by inhibiting amyloid β peptide (Aβ) aggregation and deposition. However, recent studies considered that the phenylethanoid glycosides may be metabolized by intestinal bacteria, because all analysis results showed that the bioavailability of phenylethanoid glycosides is extremely low. In this study we demonstrate how iron chelation plays a crucial role in the Aβ aggregation and deposition inhibition mechanism of phenylethanoid glycosides of CTE. In addition, we further proved phenylethanoid glycosides (1⁻3) could reach brain. Active CTE component and action mechanism confirmation will be a great help for product quality control and bioavailability studies in the future. At the same time, we provide a new analysis method useful in determining phenylethanoid glycosides (1⁻3) in plants, foods, blood, and tissues for chemical fingerprint and pharmacokinetic research.

The fibrillization and abnormal aggregation of β-amyloid (Aβ) peptides are commonly recognized risk factors for Alzheimer's disease (AD) brain, and require an effective strategy to inhibit the Aβ deposition and treat AD. Herein, we designed and synthesized nitrogen-doped carbon dots (N-CDs) as an Aβ-targeted probe, which exhibits the capacity of inhibiting the 1-42 Aβ (Aβ1-42) self-assembly in vitro. The N-CDs exhibited orange emission with an emission wavelength of 570 nm, which demonstrates their excellent optical properties with excitation-independent behavior. Meanwhile, the N-CDs have spherical morphologies with an average size of 2.2 nm, whose surface enriches the amino, carboxyl, and hydroxyl groups. These preparties are conducive to improving their biological water solubility and provide a large number of chemical bonds for further interaction with proteins. Contrary to this, the kinetic process, size evolutions, and morphologies changes of Aβ1-42 were inhibited in the presence of N-CDs in the determination of a thioflavin T assay, dynamic light scattering, transmission electron microscope, etc. Finally, the safety application of N-CDs on Aβ1-42-induced cytotoxicity was further demonstrated via in vitro cytotoxicity experiments. This work demonstrates the effective outcome of suppressing Aβ aggregation, which provides a new view into the high-efficiency and low-cytotoxicity strategy in AD theranostics.

Microcin E492 (MccE492) is an antimicrobial peptide and proposed virulence factor produced by some Klebsiella pneumoniae strains, which, under certain conditions, form amyloid fibers, leading to the loss of its antibacterial activity. Although this protein has been characterized as a model functional amyloid, the secondary structure transitions behind its formation, and the possible effect of molecules that inhibit this process, have not been investigated. In this study, we examined the ability of the green tea flavonoid epigallocatechin gallate (EGCG) to interfere with MccE492 amyloid formation. Aggregation kinetics followed by thioflavin T binding were used to monitor amyloid formation in the presence or absence of EGCG. Additionally, synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) were used to study the secondary structure, thermal stability, and morphology of microcin E492 fibers. Our results showed that EGCG significantly inhibited the formation of the MccE492 amyloid, resulting in mainly amorphous aggregates and small oligomers. However, these aggregates retained part of the β-sheet SRCD signal and a high resistance to heat denaturation, suggesting that the aggregation process is sequestered or deviated at some stage but not completely prevented. Thus, EGCG is an interesting inhibitor of the amyloid formation of MccE492 and other bacterial amyloids.

Amyloid aggregates play a major role in diseases as well as in normal physiological function. Receptor-interacting protein kinases 1 and 3 (RIP1/RIP3) aggregates complexes in cellular necroptosis is one example of protein aggregation in normal cellular function. Although recently there have been several studies on full kinase proteins aggregation, the aggregation potential of small peptide sequences of RIP1/RIP3, the physicochemical properties, and the potential in biomedical applications have not been explored. Hence, in this paper, we study the aggregation propensity of peptides consisting of four and twelve amino acid sequences in the RHIM region of RIP1/RIP3 proteins that are known to drive the beta-sheet formation and the subsequent aggregation. The aggregation kinetics, physicochemical characterization, mechanosensitive properties, cellular effects, and potential as a cancer drug depot have been investigated. The results show that the number and concentration of amino acids play a role in amyloid-like aggregates' properties. Further, the aggregates when formulated with cisplatin-induced significant lung cancer cell toxicity compared to an equal amount of cisplatin with and without ultrasound. The study would serve as a platform for further investigation on RIP1/RIP3 peptide and protein aggregates, their role in multiple cellular functions and diseases, and their potential as drug depots.

PAP248⁻286, a 39 amino acid peptide fragment, derived from the prostatic acid phosphatase secreted in human semen, forms amyloid fibrils and facilitates the attachment of retroviruses to host cells that results in the enhancement of viral infection. Therefore, the inhibition of amyloid formation by PAP248⁻286 (termed PAP f39) may likely reduce HIV transmission in AIDS. In this study, we show that the naphthoquinone tryptophan (NQTrp) hybrid molecule significantly inhibited PAP f39 aggregation in vitro in a dose-dependent manner as observed from the ThT assay, ANS assay, and transmission electron microscopy imaging. We found that even at a sub-molar concentration of 20:1 [PAP f39:NQTrp], NQTrp could reduce >50% amyloid formation. NQTrp inhibition of PAP f39 aggregation resulted in non-toxic intermediate species as determined by the vesicle leakage assay. Isothermal titration calorimetry and molecular docking revealed that the binding of NQTrp and PAP f39 is spontaneous, and NQTrp predominantly interacts with the polar and charged residues of the peptide by forming hydrogen bonds and hydrophobic contacts with a strong binding energy. Collectively, these findings indicate that NQTrp holds significant potential as a small molecule inhibitor of semen amyloids.

Alzheimer's disease (AD) is a neurodegenerative disorder with an increasing need for developing disease-modifying treatments as current therapies only provide marginal symptomatic relief. Recent evidence suggests the γ-aminobutyric acid (GABA) neurotransmitter system undergoes remodeling in AD, disrupting the excitatory/inhibitory (E/I) balance in the brain. Altered expression levels of K-Cl-2 (KCC2) and N-K-Cl-1 (NKCC1), which are cation-chloride cotransporters (CCCs), have been implicated in disrupting GABAergic activity by regulating GABAA receptor signaling polarity in several neurological disorders, but these have not yet been explored in AD. NKCC1 and KCC2 regulate intracellular chloride [Cl-]i by accumulating and extruding Cl-, respectively. Increased NKCC1 expression in mature neurons has been reported in these disease conditions, and bumetanide, an NKCC1 inhibitor, is suggested to show potential therapeutic benefits. This study used primary mouse hippocampal neurons to explore if KCC2 and NKCC1 expression levels are altered following beta-amyloid (Aβ1-42) treatment and the potential neuroprotective effects of bumetanide. KCC2 and NKCC1 expression levels were also examined in 18-months-old male C57BL/6 mice following bilateral hippocampal Aβ1-42 stereotaxic injection. No change in KCC2 and NKCC1 expression levels were observed in mouse hippocampal neurons treated with 1 nM Aβ1-42, but NKCC1 expression increased 30-days post-Aβ1-42-injection in the CA1 region of the mouse hippocampus. Primary mouse hippocampal cultures were treated with 1 nM Aβ1-42 alone or with various concentrations of bumetanide (1 µM, 10 µM, 100 µM, 1 mM) to investigate the effect of the drug on cell viability. Aβ1-42 produced 53.1 ± 1.4% cell death after 5 days, and the addition of bumetanide did not reduce this. However, the drug at all concentrations significantly reduced cell viability, suggesting bumetanide is highly neurotoxic. In summary, these results suggest that chronic exposure to Aβ1-42 alters the balance of KCC2 and NKCC1 expression in a region-and layer-specific manner in mouse hippocampal tissue; therefore, this process most likely contributes to altered hippocampal E/I balance in this model. Furthermore, bumetanide induces hippocampal neurotoxicity, thus questioning its suitability for AD therapy. Further investigations are required to examine the effects of Aβ1-42 on KCC2 and NKCC1 expression and whether targeting CCCs might offer a therapeutic approach for AD.

We present an integrated delivery technology herein employing the aerosolized method to repurpose thioflavin S for imaging amyloid beta (Abeta) deposits in the retina as a surrogate of Abeta in the brain for early detection of Alzheimer's disease. The data showed that wild type (WT) mice also have Abeta deposits in the retinae, albeit much less than 5XFAD mice. Further, only in 5XFAD mice, significant Abeta deposits were found associated with retinal ganglion cells (RGCs) in whole-mount and cross-section data. Furthermore, the fluorescent signal depicted from thioflavin S corroborates with Abeta immunohistochemistry staining information. Overall, this probe delivery via inhalation method is also applicable to other Abeta-binding molecules, such as Congo red, curcumin, and thioflavin T. The advantage of imaging retinal amyloid deposits compared to the brain counterparts is that the eye is easily accessible by in vivo imaging and it reduces the effort to design a probe that must cross the formidable blood-brain barrier.

The polyphenols curcumin (CU) and ferulic acid (FA) are able to inhibit the aggregation of amyloid-β (Aβ) peptide with different strengths. CU is a strong inhibitor while FA is a weaker one. In the present study, we examine the effects of CU and FA on the folding process of an Aβ monomer by 1 µs molecular dynamics (MD) simulations. We found that both inhibitors increase the helical propensity and decrease the non-helical propensity of Aβ peptide. They prevent the formation of a dense bulk core and shorten the average lifetime of intramolecular hydrogen bonds in Aβ. CU makes more and longer-lived hydrogen bonds, hydrophobic, π-π, and cation-π interactions with Aβ peptide than FA does, which is in a good agreement with the observed stronger inhibitory activity of CU on Aβ aggregation.