Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide.

Extracellular vesicles (EVs) carry various informative components, including signaling proteins, transcriptional regulators, lipids, and nucleic acids. These components are utilized for cell-cell communication between donor and recipient cells. EVs have shown great promise as pharmaceutical-targeting vesicles and have attracted the attention of researchers in the fields of biological and medical science because of their importance as diagnostic and prognostic markers. However, the isolation and purification of EVs from cell-cultured media remain challenging. Ultracentrifugation is the most widely used method, but it requires specialized and expensive equipment. In the present study, we proposed a novel methodology to isolate EVs using a simple and convenient method, i.e., an EV catch-and-release isolation system (EV-CaRiS) using a net-charge invertible curvature-sensing peptide (NIC). Curvature-sensing peptides recognize vesicles by binding to lipid-packing defects on highly curved membranes regardless of the expression levels of biomarkers. NIC was newly designed to reversibly capture and release EVs in a pH-dependent manner. NIC allowed us to achieve reproducible EV isolation from three human cell lines on resin using a batch method and single-particle imaging of EVs containing the ubiquitous exosome markers CD63 and CD81 by total internal reflection fluorescence microscopy (TIRFM). EV-CaRiS was demonstrated as a simple and convenient methodology for EV isolation, and NIC is promising for applications in the single-particle analysis of EVs.

High performance plasma amyloid-ß biomarkers for Alzheimer's disease.

To facilitate clinical trials of disease-modifying therapies for Alzheimer's disease, which are expected to be most efficacious at the earliest and mildest stages of the disease, supportive biomarker information is necessary. The only validated methods for identifying amyloid-ß deposition in the brain-the earliest pathological signature of Alzheimer's disease-are amyloid-ß positron-emission tomography (PET) imaging or measurement of amyloid-ß in cerebrospinal fluid. Therefore, a minimally invasive, cost-effective blood-based biomarker is desirable. Despite much effort, to our knowledge, no study has validated the clinical utility of blood-based amyloid-ß markers. Here we demonstrate the measurement of high-performance plasma amyloid-ß biomarkers by immunoprecipitation coupled with mass spectrometry. The ability of amyloid-ß precursor protein (APP)669-711/amyloid-ß (Aß)1-42 and Aß1-40/Aß1-42 ratios, and their composites, to predict individual brain amyloid-ß-positive or -negative status was determined by amyloid-ß-PET imaging and tested using two independent data sets: a discovery data set (Japan, n = 121) and a validation data set (Australia, n = 252 including 111 individuals diagnosed using 11C-labelled Pittsburgh compound-B (PIB)-PET and 141 using other ligands). Both data sets included cognitively normal individuals, individuals with mild cognitive impairment and individuals with Alzheimer's disease. All test biomarkers showed high performance when predicting brain amyloid-ß burden. In particular, the composite biomarker showed very high areas under the receiver operating characteristic curves (AUCs) in both data sets (discovery, 96.7%, n = 121 and validation, 94.1%, n = 111) with an accuracy approximately equal to 90% when using PIB-PET as a standard of truth. Furthermore, test biomarkers were correlated with amyloid-ß-PET burden and levels of Aß1-42 in cerebrospinal fluid. These results demonstrate the potential clinical utility of plasma biomarkers in predicting brain amyloid-ß burden at an individual level. These plasma biomarkers also have cost-benefit and scalability advantages over current techniques, potentially enabling broader clinical access and efficient population screening.

Effects of Gly Residue and Cholesterol on the GXXXG-Mediated Parallel Association of Transmembrane Helices: A Single-Pair FRET Study.

Small residue-mediated interhelical packing is ubiquitous in helical membrane proteins: however, the lipid dependence of its stability remains unclear. We previously demonstrated that the introduction of a GXXXG sequence in the middle of de novo-designed (AALALAA)3 helices (AALALAA AGLALGA AALALAA) facilitated their dimerization, which was abolished by cholesterol. Here single-pair FRET measurements revealed that a longer GXXXGXXXG segment (AALALAA A GLALGA AAGALAA) promoted helix dimerization in POPC/cholesterol bilayers, but not without cholesterol. The predicted dimer structures and degrees of helix packing suggested that helix dimers with small (∼10°) and large (∼55°) crossing angles were only stabilized in POPC and POPC/cholesterol membranes, respectively. A steric hindrance in the dimer interface and the large flexibility of helices prevented the formation of stable dimers. Therefore, amino acid sequences and lipid compositions distinctively constrain stable dimer structures in membranes.

Elucidation of Complex Dynamic Intermolecular Interactions in Membranes.

Biomembranes composed of various proteins and lipids play important roles in cellular functions, such as signal transduction and substance transport. In addition, some bioactive peptides and pathogenic proteins target membrane proteins and lipids to exert their effects. Therefore, an understanding of dynamic and complex intermolecular interactions among these membrane constituents is needed to elucidate their mechanisms. This review summarizes the major research carried out in the author's laboratory on how lipids and their inhomogeneous distributions regulate the structures and functions of antimicrobial peptides and Alzheimer's amyloid ß-protein. Also, how to detect transmembrane helix-helix and membrane protein-protein interactions and how they are modulated by lipids are discussed.

Meeting Peptides in Kyoto.

The 10th International Peptide Symposium was held in Kyoto last December in conjunction with the 55th Japanese Peptide Symposium. Around 800 peptide scientists from 31 different countries and regions enjoyed sessions covering various aspects of state-of-the-art peptide science, such as synthetic methodology, chemical biology, cell biology, biophysics, and medicinal/ medical applications.

Endowment of pH Responsivity to Anticancer Peptides by Introducing 2,3-Diaminopropionic Acid Residues.

Endowment of pH responsivity to anticancer peptides is a promising approach to achieve better selectivity to cancer tissues. In this research, a template peptide was designed based on magainin 2, an antimicrobial peptide with anticancer activity, and a series of peptides were designed by replacing different numbers of lysine with the unnatural amino acid, 2,3diaminopropionic acid (Dap), which has a positive charge at weakly acidic pH in cancer tissues, but is neutral at physiological pH 7.4. These Dap-containing peptides are expected to interact more strongly with tumor cells than with normal cells because 1) weakly acidic conditions form in tumors, and 2) the membrane of tumor cells is more anionic than that of normal cells. Although all examined peptides showed potent cytotoxicities to multidrug-resistant cancer cells at a weakly acidic pH (ED50 ≈5 µm), the toxicity decreased with an increase in the number of Dap at pH 7.4 (8 Dap residues resulted in ED50 ≈60 µm). Furthermore, the introduction of Dap reduced cytotoxicity against normal cells. Thus, Dap led to significantly improved cancer targeting due to a pH-dependent charge shift. Fluorescence imaging and model membrane experiments supported this charge-shift model.

Membrane Permeabilization Mechanisms.

Many antimicrobial peptides are considered to kill microbes by permeabilizing cell membranes. This chapter summarizes the driving force of peptide binding to membranes; various mechanisms of lipid bilayer permeabilization including the barrel-stave, toroidal pore, and carpet models; and modes of permeabilization of bacterial and mammalian membranes.

Computational Study on the Assembly of Amyloid ß-Peptides in the Hydrophobic Environment.

Fibrillated aggregation of amyloid ß (Aß) peptides is a potential factor causing toxic amyloid deposition in neurodegenerative diseases. A toxic fibril formation of Aß is known to be enhanced on the ganglioside-rich lipid membrane containing some amounts of cholesterol and sphingomyelin. This ganglioside-rich membrane is supposed to provide a hydrophobic environment that promotes the formation of Aß fibrils. Molecular dynamics simulations were carried out to investigate the structure of Aß complex in the hydrophobic solution composed of dioxane and water molecules. The Aß conformation was contrasted to that in the aqueous condition by executing multiple computational trials with the calculation models containing one, four, or six Aß peptides. The conformation was also compared between the calculations with the 42-mer (Aß42) and 40-mer (Aß40) peptides. The simulations for Aß42 demonstrated that Aß peptides had a tendency to stretch out in the hydrophobic environment. In contrast, Aß peptides were closely packed in the aqueous solution, and the motions of Aß peptides were suppressed significantly. The N-terminal polar domains of Aß peptides tended to be positioned at the inside of the Aß complex in the hydrophobic environment, which supported the C-terminal domains in expanding outward for inter-molecular interaction. Since Aß peptides were not tightly packed in the hydrophobic environment, the total surface area of the Aß complex in the hydrophobic solution was larger than that in the aqueous one. The simulation for Aß40 peptides also showed a difference between the hydrophobic and aqueous solutions. The difference was compatible with the results of Aß42 in the structure of the Aß complex, while the C-terminal outward expansion was not so distinct as Aß42 peptides.

Not Oligomers but Amyloids are Cytotoxic in the Membrane-Mediated Amyloidogenesis of Amyloid-ß Peptides.

The formation of neurotoxic aggregates by amyloid-ß peptide (Aß) is considered to be a key step in the onset of Alzheimer's disease. It is widely accepted that oligomers are more neurotoxic than amyloid fibrils in the aqueous-phase aggregation of Aß. Membrane-mediated amyloidogenesis is also relevant to the pathology, although the relationship between the aggregate size and cytotoxicity has remained elusive. Here, aggregation processes of Aß on living cells and cytotoxic events were monitored by fluorescence techniques. Aß formed amyloids after forming oligomers composed of ≈10 Aß molecules. The formation of amyloids was necessary to activate apoptotic caspase-3 and reduce the ability of the cell to proliferate; this indicated that amyloid formation is a key event in Aß-induced cytotoxicity.

Simulation Study on Complex Conformations of Aß42 Peptides on a GM1 Ganglioside-Containing Lipid Membrane.

Aggregation and complex formation of amyloid beta (Aß) peptides on a neuronal cell membrane is a hallmark of neuro-disturbance diseases. In this work, we performed molecular dynamics (MD) simulations to investigate the initial stage of interactions of multiple Aß42 peptides on a GM1 ganglioside-containing membrane that mimics a micro-domain on the neuronal cell surface. Conformational changes of Aßs due to adhesion on the membrane and subsequent molecular interactions among the Aßs were monitored. It was suggested from results of the two 1.0 µs simulation trials that stable complexes of Aß peptides were not rapidly generated but that a steady binding of two Aßs was gradually formed. Observation of two Aßs that will be a complex with steady binding revealed that one Aß was bound to the membrane surface, while the other was attached to the first one without strong contact with the membrane. The motion of the first one was restricted and its conformational change was limited, with the basic side-chains of Arg5 and Lys28 working as anchors to hold the Aß helix region on the membrane. In contrast, the second one had high flexibility and showed diversity in its conformation. The second Aß can search for an energetically favorable binding position on the first one. A parallel ß-sheet structure was formed between the C-terminal sides of the two Aßs. Ala30 was critically important to lead the stable ß-sheet conformation at the C-terminal hydrophobic domains of Aßs. In the N-terminal sides, helix structures were kept in both Aßs.

A pH-dependent charge reversal peptide for cancer targeting.

Naturally occurring cationic antimicrobial peptides exhibit not only antimicrobial activity, but also anticancer activity and are expected to be new weapons in cancer treatment. The selectivity for cancer cells over normal cells is at least partly due to the more negative surface of cancer cells. A lower pH in tumor tissue (pH 6.2-6.9) than that in normal tissues (pH 7.3-7.4) has also been utilized to develop anticancer agents. However, cytotoxicity against normal cells at physiological pH is often an issue. Furthermore, acidic regions can be found in some normal tissues such as the kidneys. Therefore, existing approaches to cancer targeting are not fully satisfactory. In this study, we designed a peptide, HE (GIHHWLHSAHEFGEHFVHHIMNS-amide), with a charge that reverses from -1.5 at pH 7.4 to +6 at pH 5.5 for cancer targeting at low pH based on the antimicrobial peptide magainin 2 by introducing 6 His, an additional Glu, and an amidated terminal. HE interacted with cancer-mimicking negatively charged liposomes in a pH-dependent fashion with a midpoint with a pH of 6.5 just above the membrane surface. The peptide killed human renal adenocarcinoma ACHN cells at pH 6.0, but not at pH 7.4, and was nontoxic against human normal glomerular mesangial cells even at this low pH. Thus, the novel peptide may be a promising lead peptide for cancer therapy, although this derivatization resulted in weakened cytotoxicity.

Stoichiometric analysis of oligomeric states of three class-A GPCRs, chemokine-CXCR4, dopamine-D2, and prostaglandin-EP1 receptors, on living cells.

G-protein-coupled receptors (GPCRs) form the largest family of transmembrane receptors, and their oligomerization has been suggested to be related to their functions. Despite extensive studies, their oligomeric states are highly controversial. One of the reasons is the overestimation of oligomerization by conventional methods. We recently established a stoichiometric analysis method for precisely determining the oligomeric state of membrane proteins on living cells with the combined use of the coiled-coil labeling method and a spectral imaging technique and showed that the prototypical class-A GPCR ß2 -adrenergic receptor (ß2 AR) did not form functional oligomers. In this study, we expanded our study to three well-studied class-A GPCRs: C-X-C chemokine receptor of stromal cell-derived factor-1α (CXCR4), dopamine receptor D2 short isotype (D2R), and prostaglandin E receptor subtype 1 (EP1R). We found that these receptors did not form constitutive homooligomers. The receptors exhibited calcium signaling upon agonist stimulation as monomers, although CXCR4 and EP1R gradually clustered after fast signaling. We conclude that homooligomerization is not necessary for the signal transductions of these four class-A GPCRs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Aromaticity of Phenylalanine Residues Is Essential for Amyloid Formation by Alzheimer's Amyloid ß-Peptide.

The abnormal aggregation of amyloid ß-peptide (Aß) is central to the pathogenesis of Alzheimer's disease, the major form of dementia. Aromatic π-π interactions have been suggested to play a crucial role in the aggregation of not only Aß, but also other amyloidogenic proteins. In this study, each or all phenylalanine (Phe) residues at the 4th, 19th, and 20th positions of Aß-(1-40) were substituted by hydrophobic cyclohexylalanine (Cha), which is sterically similar to Phe, but lacks π-electrons, to reveal effects of interactions involving π-electrons on the aggregation of Aß both in aqueous solution and GM1-containing membranes. We found that each Cha substitution significantly inhibited fibril formation by Aß, indicating a pivotal role of aromatic interactions. Furthermore, the Aß analog with three Cha residues effectively retarded the fibrillation of the wild-type Aß.

GXXXG-Mediated Parallel and Antiparallel Dimerization of Transmembrane Helices and Its Inhibition by Cholesterol: Single-Pair FRET and 2D IR Studies.

Small-residue-mediated interhelical packings are ubiquitously found in helical membrane proteins, although their interaction dynamics and lipid dependence remain mostly uncharacterized. We used a single-pair FRET technique to examine the effect of a GXXXG motif on the association of de novo designed (AALALAA)3 helices in liposomes. Dimerization occurred with sub-second lifetimes, which was abolished by cholesterol. Utilizing the nearly instantaneous time-resolution of 2D IR spectroscopy, parallel and antiparallel helix associations were identified by vibrational couplings across helices at their interface. Taken together, the data illustrate that the GXXXG motif controls helix packing but still allows for a dynamic and lipid-regulated oligomeric state.

Effects of C-terminal Truncation of Chaperonin GroEL on the Yield of In-cage Folding of the Green Fluorescent Protein.

Chaperonin GroEL from Escherichia coli consists of two heptameric rings stacked back-to-back to form a cagelike structure. It assists in the folding of substrate proteins in concert with the co-chaperonin GroES by incorporating them into its large cavity. The mechanism underlying the incorporation of substrate proteins currently remains unclear. The flexible C-terminal residues of GroEL, which are invisible in the x-ray crystal structure, have recently been suggested to play a key role in the efficient encapsulation of substrates. These C-terminal regions have also been suggested to separate the double rings of GroEL at the bottom of the cavity. To elucidate the role of the C-terminal regions of GroEL on the efficient encapsulation of substrate proteins, we herein investigated the effects of C-terminal truncation on GroE-mediated folding using the green fluorescent protein (GFP) as a substrate. We demonstrated that the yield of in-cage folding mediated by a single ring GroEL (SR1) was markedly decreased by truncation, whereas that mediated by a double ring football-shaped complex was not affected. These results suggest that the C-terminal region of GroEL functions as a barrier between rings, preventing the leakage of GFP through the bottom space of the cage. We also found that once GFP folded into its native conformation within the cavity of SR1 it never escaped even in the absence of the C-terminal tails. This suggests that GFP molecules escaped through the pore only when they adopted a denatured conformation. Therefore, the folding and escape of GFP from C-terminally truncated SR1·GroES appeared to be competing with each other.

Oligomerization-function relationship of EGFR on living cells detected by the coiled-coil labeling and FRET microscopy.

The epidermal growth factor receptor (EGFR) is a well-studied receptor tyrosine kinase and an important anticancer therapeutic target. The activity of EGFR autophosphorylation and transphosphorylation, which induces several cell signaling pathways, has been suggested to be related to its oligomeric state. However, the oligomeric states of EGFRs induced by EGF binding and the receptor-ligand stoichiometry required for its activation are still controversial. In the present study, we performed Förster resonance energy transfer (FRET) measurements by combining the coiled-coil tag-probe labeling method and spectral imaging to quantitatively analyze EGFR oligomerization on living CHO-K1 cell membranes at physiological expression levels. In the absence of its ligands, EGFRs mainly existed as monomers with a small fraction of predimers (~10%), whereas ~70% of the EGFRs formed dimers after being stimulated with the ligand EGF. Ligand-induced dimerization was not significantly affected by the perturbation of membrane components (cholesterol or monosialoganglioside GM3). We also investigated both dose and time dependences of EGF-dependent EGFR dimerization and autophosphorylation. The formation of dimers occurred within 20s of the ligand stimulation and preceded its autophosphorylation, which reached a plateau 90 s after the stimulation. The EGF concentration needed to evoke half-maximum dimerization (~1 nM) was lower than that for half-maximum autophosphorylation (~8 nM), which suggested the presence of an inactive dimer binding a single EGF molecule.

Selective amine labeling of cell surface proteins guided by coiled-coil assembly.

Covalent labeling of target proteins in living cells is useful for both fluorescence live-cell imaging and the subsequent biochemical analyses of the proteins. Here, we report an efficient method for the amine labeling of membrane proteins on the cell surface, guided by a noncovalent coiled-coil interaction. A carboxyl sulfosuccinimidyl ester introduced at the C-terminus of the coiled-coil probe reacted with target proteins under mild labeling conditions ([probe] = 150 nM, pH 7.4, 25°C) for 20 min. Various fluorescent moieties with different hydrophobicities are available for covalent labeling with high signal/background labeling ratios. Using this method, oligomeric states of glycophorin A (GpA) were compared in mammalian CHO-K1 cells and sodium dodecyl sulfate (SDS) micelles. In the cell membranes, no significant self-association of GpA was detected, whereas SDS-PAGE suggested partial dimerization of the proteins. Membrane cholesterol was found to be an important factor that suppressed the dimerization of GpA. Thus, the covalent functionality enables direct comparison of the oligomeric state of membrane proteins under various conditions. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 484-490, 2016.

Cholesterol-induced lipophobic interaction between transmembrane helices using ensemble and single-molecule fluorescence resonance energy transfer.

The solvent environment regulates the conformational dynamics and functions of solvated proteins. In cell membranes, cholesterol, a major eukaryotic lipid, can markedly modulate protein dynamics. To investigate the nonspecific effects of cholesterol on the dynamics and stability of helical membrane proteins, we monitored association-dissociation dynamics on the antiparallel dimer formation of two simple transmembrane helices (AALALAA)3 with single-molecule fluorescence resonance energy transfer (FRET) using Cy3B- and Cy5-labeled helices in lipid vesicles (time resolution of 17 ms). The incorporation of 30 mol % cholesterol into phosphatidylcholine bilayers significantly stabilized the helix dimer with average lifetimes of 450-170 ms in 20-35 °C. Ensemble FRET measurements performed at 15-55 °C confirmed the cholesterol-induced stabilization of the dimer (at 25 °C, ΔΔG(a) = -9 kJ mol(-1) and ΔΔHa = -60 kJ mol(-1)), most of which originated from "lipophobic" interactions by reducing helix-lipid contacts and the lateral pressure in the hydrocarbon core region. The temperature dependence of the dissociation process (activation energy of 48 kJ) was explained by the Kramers-type frictional barrier in membranes without assuming an enthalpically unfavorable transition state. In addition to these observations, cholesterol-induced tilting of the helices, a positive ΔC(p(a)), and slower dimer formation compared with the random collision rate were consistent with a hypothetical model in which cholesterol stabilizes the helix dimer into an hourglass shape to relieve the lateral pressure. Thus, the liposomal single-molecule approach highlighted the significance of the cholesterol-induced basal force for interhelical interactions, which will aid discussions of complex protein-membrane systems.

How do membranes initiate Alzheimer's Disease? Formation of toxic amyloid fibrils by the amyloid ß-protein on ganglioside clusters.

Alzheimer's disease (AD), a severe neurodegenerative disorder, causes more than half of dementia cases. According to the popular "Aß hypothesis" to explain the mechanism of this disease, amyloid ß-peptides (Aß) of 39-43 amino acid residues aggregate and deposit onto neurons, igniting the neurotoxic cascade of the disease. Therefore, researchers studying AD would like to elucidate the mechanisms by which essentially water-soluble but hydrophobic Aß aggregates under pathological conditions. Most researchers have investigated the aggregation of Aß in aqueous solution, and they concluded that the final aggregation product, the amyloid fibrils, were less toxic than the component peptide oligomers. They consequently shifted their interests to more toxic "soluble oligomers", structures that form as intermediates or off-pathway products during the aggregation process. Some researchers have also investigated artificial oligomers prepared under nonphysiological conditions. In contrast to these "in solution" studies, we have focused on "membrane-mediated" amyloidogenesis. In an earlier study, other researchers identified a specific form of Aß that was bound to monosialoganglioside GM1, a sugar lipid, in brains of patients who exhibited the early pathological changes associated with AD. This Account summarizes 15 years of our research on this topic. We have found that Aß specifically binds to GM1 that occurs in clusters, but not when it is uniformly distributed. Clustering is facilitated by cholesterol. Upon binding, Aß changes its conformation from a random coil to an α-helix-rich structure. A CH-π interaction between the aromatic side chains of Aß and carbohydrate moieties appended to GM1 appears to be important for binding. In addition, as Aß accumulates and reaches its first threshold concentration (Aß/GM1 = ∼0.013), aggregated ß-sheets of ∼15 molecules appear and coexist with the helical form. However, this ß-structure is stable and does not form larger aggregates. When the disease progresses further and the Aß/GM1 ratio exceeds ∼0.044, the ß-structure converts to a second ß-structure that can seed aggregates. The seed recruits monomers from the aqueous phase to form toxic amyloid fibrils that have larger surface hydrophobicity and can contain antiparallel ß-sheets. In contrast, amyloid fibrils formed in aqueous solution are less toxic and have parallel ß-sheets. The less polar environments of GM1 clusters play an important role in the formation of these toxic fibrils. Membranes that contain GM1 clusters not only accelerate the aggregation of Aß by locally concentrating Aß molecules but also generate amyloid fibrils with unique structures and significant cytotoxicity. The inhibition of this aggregation cascade could be a promising strategy for the development of AD-modulating therapies.

Comparison between the aggregation of human and rodent amyloid ß-proteins in GM1 ganglioside clusters.

The abnormal deposition of amyloids by amyloid-ß protein (Aß) is a pathological hallmark of Alzheimer's disease (AD). Aged rodents rarely develop the characteristic lesions of the disease, which is different from the case in humans. Rodent Aß (rAß) differs from human Aß (hAß) only in the three substitutions of Arg to Gly, Tyr to Phe, and His to Arg at positions 5, 10, and 13, respectively. Understanding the reason why rodent Aß does not form amyloids is important to revealing factors that cause the abnormal aggregation of Aß under pathologic conditions. We have proposed that the binding of Aß to membranes with ganglioside clusters plays an important role in the abnormal aggregation of Aß. In this study, we compared hAß and rAß in terms of aggregation on neuronal cells, on raftlike model membranes, and in buffer. We found that rAß formed amyloid fibrils similar to those of hAß in buffer solution. In contrast, on cell membranes and raftlike membranes, hAß formed toxic, mature amyloid fibrils, whereas rAß produced less toxic protofibrils that were not stained by the amyloid-specific dye Congo red. Thus, our ganglioside cluster-mediated amyloidogenesis hypothesis explains the immunity of rodents from cerebral Aß amyloid deposition, strengthening the importance of ganglioside clusters as a platform of abnormal Aß deposition in the pathology of AD.