RESUMEN
Gene therapy is emerging as a powerful tool to modulate abnormal gene expression, a hallmark of most CNS disorders. The transformative potentials of recently approved gene therapies for the treatment of spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and active cerebral adrenoleukodystrophy are encouraging further development of this approach. However, most attempts to translate gene therapy to the clinic have failed to make it to market. There is an urgent need not only to tailor the genes that are targeted to the pathology of interest but to also address delivery challenges and thereby maximize the utility of genetic tools. In this Review, we provide an overview of gene therapy modalities for CNS diseases, emphasizing the interconnectedness of different delivery strategies and routes of administration. Important gaps in understanding that could accelerate the clinical translatability of CNS genetic interventions are addressed, and we present lessons learned from failed clinical trials that may guide the future development of gene therapies for the treatment and management of CNS disorders.
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Enfermedades del Sistema Nervioso Central , Terapia Genética , Humanos , Terapia Genética/métodos , Terapia Genética/tendencias , Enfermedades del Sistema Nervioso Central/terapia , Enfermedades del Sistema Nervioso Central/genética , Animales , Investigación Biomédica Traslacional/métodos , Técnicas de Transferencia de Gen/tendenciasRESUMEN
Lateral flow assays (LFAs) have emerged as indispensable tools for point-of-care testing during the pandemic era. However, the interpretation of results through unassisted visual inspection by untrained individuals poses inherent limitations. In our study, we propose a novel approach that combines computer vision (CV) and lightweight machine learning (ML) to overcome these limitations and significantly enhance the performance of LFAs. By incorporating CV-assisted analysis into the LFA assay, we achieved a remarkable three-fold improvement in analytical sensitivity for detecting Influenza A and for SARS-CoV-2 detection. The obtained R2 values reached approximately 0.95, respectively, demonstrating the effectiveness of our approach. Moreover, the integration of CV techniques with LFAs resulted in a substantial amplification of the colorimetric signal specifically for COVID-19 positive patient samples. Our proposed approach, which incorporates a simple machine learning algorithm, provides substantial enhancements in assay sensitivity, improving diagnostic efficacy and accessibility of point-of-care testing without requiring significant additional resources. Moreover, the simplicity of the machine learning algorithm enables its standalone use on a mobile phone, further enhancing its practicality for point-of-care testing.
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COVID-19 , Gripe Humana , Humanos , SARS-CoV-2 , Gripe Humana/diagnóstico , COVID-19/diagnóstico , Algoritmos , Bioensayo , Prueba de COVID-19RESUMEN
Colorimetric glucose sensors using enzyme-coronated gold nanoparticles have been developed for high-throughput assays to monitor the blood glucose levels of diabetic patients. Although those sensors have shown sensitivity and wide linear detection ranges, they suffer from poor selectivity and stability in detecting blood glucose, which has limited their practical use. To address this limitation, herein, we functionalized glucose-oxidase-coronated gold nanoparticles with an erythrocyte membrane (EM-GOx-GNPs). Because the erythrocyte membrane (EM) selectively facilitates the permeation of glucose via glucose transporter-1 (GLUT1), the functionalization of GOx-GNPs with EM improved the stability, selectivity (3.3- to 15.8-fold higher), and limit of detection (LOD). Both membrane proteins, GLUT1 and aquaporin-1 (AQP1), on EM were shown to be key components for selective glucose detection by treatment with their inhibitors. Moreover, we demonstrated the stability of EM-GOx-GNPs in high-antioxidant-concentration conditions, under long-term storage (â¼4 weeks) and a freeze-thaw cycle. Selectivity of the EM-GOx-GNPs against other saccharides was increased, which improved the LOD in phosphate-buffered saline and human serum. Our results indicated that the functionalization of colorimetric glucose sensors with EM is beneficial for improving selectivity and stability, which may make them candidates for use in a practical glucose sensor.
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Técnicas Biosensibles , Nanopartículas del Metal , Técnicas Biosensibles/métodos , Glucemia , Membrana Eritrocítica , Glucosa , Glucosa Oxidasa/metabolismo , Transportador de Glucosa de Tipo 1 , Oro/metabolismo , HumanosRESUMEN
In the development of enzymatic glucose sensors, accurate glucose sensing has been a challenging task because of the existence of numerous interfering molecules in the blood. Meanwhile, red blood cells (RBCs) selectively uptake glucose via a membrane protein called glucose transporter-1. In this study, we developed the RBC membrane (RBCM)-coated enzymatic glucose sensors that mimic the glucose uptake. The RBCM-coated sensors were examined via scanning electron microscopy, atomic force microscopy, and ATR-FTIR. We optimized the glucose permeability of the RBCM filter by controlling the thickness of the filter. The sensing range of the optimized sensor was 1-15 mM, the detection limit was 0.66 mM, and the sensitivity was 2.978 µA mM-1. Intriguingly, the RBCM-coated sensor was highly accurate and precise, despite the coexistence of glucose and interfering molecules (e.g., mannose, galactose, ascorbic acid, uric acid, and cysteine). For each interfering molecule, the errors of our sensor were 0.8 to 2.3%, which was 4.8-14.2 times more accurate than the uncoated one. A similar result was verified for a human serum sample containing countless interfering molecules. Also, the sensing performance of the sensor was consistent after 4 weeks of storage. The results suggest that applying RBCM may improve the selectivity of various types of glucose sensors including the continuous monitoring system.
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Glucemia/análisis , Técnicas Electroquímicas/métodos , Membrana Eritrocítica/química , Eritrocitos/química , Glucosa 1-Deshidrogenasa/química , Glucemia/química , Técnicas Electroquímicas/instrumentación , Electrodos , Enzimas Inmovilizadas/química , Transportador de Glucosa de Tipo 1/química , Humanos , Oxidación-ReducciónRESUMEN
The surface potential of nanoparticles plays a key role in numerous applications, such as drug delivery and cellular uptake. The estimation of the surface potential of nanoparticles as drug carriers or contrast agents is important for the design of nanoparticle-based biomedical platforms. Herein, we report the direct measurement of the surface potential of individual gold nanorods (GNRs) via Kelvin probe force microscopy (KPFM) at the nanoscale. GNRs were capped by a surfactant, cetyltrimethylammonium bromide (CTAB), which was removed by centrifugation. CTAB removal is essential for GNR-based biomedical applications because of the cytotoxicity of CTAB. Applying KPFM analysis, we found that the mean surface potential of the GNRs became more negative as the CTAB was removed from the GNR. The results indicate that the negative charge of GNRs is covered by the electrostatic charge of the CTAB molecules. Similar trends were observed in experiments with gold nanospheres (GNS) capped by citrates. Overall, KPFM-based techniques characterize the surfactant of individual nanoparticles (i.e. GNR or GNS) with high resolution by mapping the surface potential of a single nanoparticle, which aids in designing engineered nanoparticles for biomedical applications.
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With the rapid development of nanotechnology and its associated waste stream, public concern is growing over the potential toxicity exposure to heavy metal ions poses to the human body and the environment. Herein, we report an extremely sensitive Kelvin probe force microscopy (KPFM)-based platform for detecting nanotoxic materials (e.g. Ag+) accomplished by probing the integrated surface potential differences of a single gold nanoparticle on which an interaction between probe DNA and target DNA occurs. This interaction can amplify the surface potential of the nanoparticle owing to the coordination bond mediated by Ag+ (cytosine-Ag+-cytosine base pairs). Interestingly, compared with conventional methods, this platform is capable of extremely sensitive Ag+ detection (â¼1 fM) in a remarkably wide-range (1 fM to 1 µM). Furthermore, this platform enables Ag+ detection in a practical sample (general drinking water), and this KPFM-based technique may have the potential to detect other toxic heavy metal ions and single nucleotide polymorphisms by designing specific DNA sequences.
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ADN/química , Agua Potable/análisis , Oro/química , Nanopartículas del Metal/química , Plata/análisis , Contaminantes Químicos del Agua/análisis , Sondas de ADN/química , Límite de Detección , Microscopía de Fuerza Atómica/métodosRESUMEN
Alzheimer's disease (AD) is the most common cause of neurodegenerative disorder in elderly people, and has become a social problem in aging societies globally. Amyloid-ß (Aß) aggregates (i.e., Aß fibrils and plaques) present in the brains of AD patients are hallmarks of AD. Although various promising anti-Aß drugs have been tested in pre-clinical and randomized controlled trials, the trial results have not yet been translated into clinical practice due to increasing time and cost of drug development. Recent investigations have addressed how the formation of Aß aggregates is influenced by the surface of gold nanoparticles (AuNPs) to obtain a detailed understanding of the in vivo process of amyloid formation. Particularly, AuNPs catalytically provide nucleation sites to accelerate the formation of Aß aggregates. Moreover, AuNPs have great potential as a sensing tool due to their optical property. Employing this dual function (i.e., catalytic and optical property), AuNP-based colorimetry is highlighted as a simple and innovative method for monitoring the efficacy of anti-Aß reagents. In this review, we briefly survey important developments and designs of anti-Aß drugs. The significance and perspectives of AuNP-based drug-screening in pharmacologic research are also discussed.
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Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides , Nanopartículas del Metal , Evaluación de Medicamentos , Oro , Humanos , Placa AmiloideRESUMEN
Amyloid aggregates have emerged as a significant hallmark of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Although it has been recently reported that microwave heating induces amyloid aggregation compared with conventional heating methods, the mechanism of amyloid aggregate induction has remained unclear. In this study, we investigated the formation of oligomeric amyloid aggregates (OAAs) by microwave irradiation at microscale volumes of solution. Microwave irradiation of protein monomer solution triggered rapid formation of OAAs within 7 min. We characterized the formation of OAAs using atomic force microscopy, thioflavin T fluorescent assay and circular dichroism. In the microwave system, we also investigated the inhibitory effect on the formation of amyloid aggregates by L-ascorbic acid as well as enhanced amyloid aggregation by silver nanomaterials such as nanoparticles and nanowires. We believe that microwave technology has the potential to facilitate the study of amyloid aggregation in the presence of chemical agents or nanomaterials.
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Amiloide/química , Microondas , Agregado de Proteínas , Dicroismo Circular , Lactoglobulinas/química , Nanoestructuras/química , Nanoestructuras/ultraestructura , Plata/químicaRESUMEN
INTRODUCTION: Intravesical drug delivery (IDD) has gained recognition as a viable approach for treating bladder-related diseases over the years. However, it comes with its set of challenges, including voiding difficulties and limitations in mucosal and epithelial penetration. These challenges lead to drug dilution and clearance, resulting in poor efficacy. Various strategies for drug delivery have been devised to overcome these issues, all aimed at optimizing drug delivery. Nevertheless, there has been minimal translation to clinical settings. AREAS COVERED: This review provides a detailed description of IDD, including its history, advantages, and challenges. It also explores the physical barriers encountered in IDD, such as voiding, mucosal penetration, and epithelial penetration, and discusses current strategies for overcoming these challenges. Additionally, it offers a comprehensive roadmap for advancing IDD into clinical trials. EXPERT OPINION: Physical bladder barriers and limitations of conventional treatments result in unsatisfactory efficacy against bladder diseases. Nevertheless, substantial recent efforts in this field have led to significant progress in overcoming these challenges and have raised important attributes for an optimal IDD system. However, there is still a lack of well-defined steps in the workflow to optimize the IDD system for clinical settings, and further research is required to establish more comprehensive in vitro and in vivo models to expedite clinical translation.
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Sistemas de Liberación de Medicamentos , Vejiga Urinaria , Administración Intravesical , Preparaciones FarmacéuticasRESUMEN
The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has evoked a worldwide pandemic. As the emergence of variants has hampered the neutralization capacity of currently available vaccines, developing effective antiviral therapeutics against SARS-CoV-2 and its variants becomes a significant challenge. The main protease (Mpro) of SARS-CoV-2 has received increased attention as an attractive pharmaceutical target because of its pivotal role in viral replication and proliferation. Here, we generated a de novo Mpro-inhibitor screening platform to evaluate the efficacies of Mpro inhibitors based on Mpro cleavage site-embedded amyloid peptide (MCAP)-coated gold nanoparticles (MCAP-AuNPs). We fabricated MCAPs comprising an amyloid-forming sequence and Mpro-cleavage sequence, mimicking in vivo viral replication process mediated by Mpro. By measuring the proteolytic activity of Mpro and the inhibitory efficacies of various drugs, we confirmed that the MCAP-AuNP-based platform was suitable for rapid screening potential of Mpro inhibitors. These results demonstrated that our MCAP-AuNP-based platform has great potential for discovering Mpro inhibitors and may accelerate the development of therapeutics against COVID-19.
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COVID-19 , Nanopartículas del Metal , Humanos , SARS-CoV-2 , Oro/farmacología , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales , Péptidos , Péptido Hidrolasas , Antivirales/farmacología , Simulación del Acoplamiento MolecularRESUMEN
Hydroxyapatite (HA) exhibits outstanding biocompatibility, bioactivity, osteoconductivity, and natural anti-inflammatory properties. Pure HA, ion-doped HA, and HA-polymer composites are investigated, but critical limitations such as brittleness remain; numerous efforts are being made to address them. Herein, the novel self-crystallization of a polymeric single-stranded deoxyribonucleic acid (ssDNA) without additional phosphate ions for synthesizing deoxyribonucleic apatite (DNApatite) is presented. The synthesized DNApatite, DNA1Ca2.2(PO4)1.3OH2.1, has a repetitive dual phase of inorganic HA crystals and amorphous organic ssDNA at the sub-nm scale, forming nanorods. Its mechanical properties, including toughness and elasticity, are significantly enhanced compared with those of HA nanorod, with a Young's modulus similar to that of natural bone.
RESUMEN
Proteolysis of amyloids is related to prevention and treatment of amyloidosis. What if the conditions for proteolysis were the same to those for amyloid formation? For example, pepsin, a gastric protease is activated in an acidic environment, which, interestingly, is also a condition that induces the amyloid formation. Here, we investigate the competition reactions between proteolysis and synthesis of amyloid under pepsin-activated conditions. The changes in the quantities and nanomechanical properties of amyloids after pepsin treatment were examined by fluorescence assay, circular dichroism and atomic force microscopy. We found that, in the case of pepsin-resistant amyloid, a secondary reaction can be accelerated, thereby proliferating amyloids. Moreover, after this reaction, the amyloid became 32.4 % thicker and 24.2 % stiffer than the original one. Our results suggest a new insight into the proteolysis-driven proliferation and rigidification of pepsin-resistant amyloids.
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Amiloide , Pepsina A , Proteolisis , Pepsina A/metabolismo , Amiloide/metabolismo , Péptido Hidrolasas/metabolismo , Dicroismo Circular , Proteínas Amiloidogénicas , Proliferación Celular , Microscopía de Fuerza AtómicaRESUMEN
Cholera is a highly contagious and lethal waterborne disease induced by an infection with Vibrio cholerae (V. cholerae) secreting cholera toxin (CTx). Cholera toxin subunit B (CTxB) from the CTx specifically binds with monosialo-tetra-hexosyl-ganglioside (GM1) found on the exterior cell membrane of an enterocyte. Bioinspired by the pathological process of CTx, we developed an electrochemical biosensor with GM1-expressing Caco-2 cell membrane (CCM) on the electrode surface. Briefly, the electrode surface was functionalized with CCM using the vesicle fusion method. We determined the CTxB detection performances of Caco-2 cell membrane-coated biosensor (CCB) using electrochemical impedance spectroscopy (EIS). the CCB had an excellent limit of detection of â¼11.46 nM and a detection range spanning 100 ng/mL - 1 mg/mL. In addition, the CCB showed high selectivity against various interfering molecules, including abundant constituents of intestinal fluid and various bacterial toxins. The long-term stability of the CCBs was also verified for 3 weeks using EIS. Overall, the CCB has excellent potential for practical use such as point-of-care and cost-effective testing for CTxB detection in developing countries.
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Técnicas Biosensibles , Cólera , Humanos , Cólera/microbiología , Toxina del Cólera , Células CACO-2 , Gangliósido G(M1) , BiomiméticaRESUMEN
The assembly of α-synuclein (αS) oligomers is recognized as the main pathological driver of synucleinopathies. While the elimination of toxic αS oligomers shows promise for the treatment of Parkinson's disease (PD), the discovery of αS oligomer degradation drugs has been hindered by the lack of proper drug screening tools. Here, we report a drug screening platform for monitoring the efficacy of αS-oligomer-degrading drugs using amyloid-shelled gold nanocomplexes (ASGNs). We fabricate ASGNs in the presence of dopamine, mimicking the in vivo generation process of pathological αS oligomers. To test our platform, the first of its kind for PD drugs, we use αS-degrading proteases and various small molecular substances that have shown efficacy in PD treatment. We demonstrate that the ASGN-based in vitro platform has strong potential to discover effective αS-oligomer-targeting drugs, and thus it may reduce the attrition problem in drug discovery for PD treatment.
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Enfermedad de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/metabolismo , Amiloide/metabolismo , Proteínas AmiloidogénicasRESUMEN
Highly sensitive rapid testing for COVID-19 is essential for minimizing virus transmission, especially before the onset of symptoms and in asymptomatic cases. Here, we report bioengineered enrichment tools for lateral flow assays (LFAs) with enhanced sensitivity and specificity (BEETLES2), achieving enrichment of SARS-CoV-2 viruses, nucleocapsid (N) proteins and immunoglobulin G (IgG) with 3-minute operation. The limit of detection is improved up to 20-fold. We apply this method to clinical samples, including 83% with either intermediate (35%) or low viral loads (48%), collected from 62 individuals (n = 42 for positive and n = 20 for healthy controls). We observe diagnostic sensitivity, specificity, and accuracy of 88.1%, 100%, and 91.9%, respectively, compared with commercial LFAs alone achieving 14.29%, 100%, and 41.94%, respectively. BEETLES2, with permselectivity and tunability, can enrich the SARS-CoV-2 virus, N proteins, and IgG in the nasopharyngeal/oropharyngeal swab, saliva, and blood serum, enabling reliable and sensitive point-of-care testing, facilitating fast early diagnosis.
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COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Prueba de COVID-19 , Sensibilidad y Especificidad , Reacción en Cadena de la Polimerasa , Inmunoglobulina GRESUMEN
For decades, acetylcholine (Ach) has been considered a critical biomarker for several degenerative brain diseases, including Alzheimer's, Parkinson's disease, Huntington's disease, and schizophrenia. Here, we propose a wafer-scale fabrication of polyaniline (PAni)-grafted graphene-based field-effect transistors (PGFET) and their biosensing applications for highly sensitive and reliable real-time monitoring of Ach in flow configuration. The grafted PAni provides suitable electrostatic binding sites for enzyme immobilization and enhances the pH sensitivity (2.68%/pH), compared to that of bare graphene-FET (1.81%/pH) for a pH range of 3-9 without any pH-hysteresis. We further evaluated the PGFET's sensing performance for Ach detection with a limit of detection at the nanomolar level and significantly improved sensitivity (~103%) in the concentration range of 108 nM to 2 mM. Moreover, the PGFET exhibits excellent selectivity against various interferences, including glucose, ascorbic acid, and neurotransmitters dopamine and serotonin. Finally, we investigated the effects of an inhibitor (rivastigmine) on the AchE activity of the PGFET. From the results, we demonstrated that the PGFET has great potential as a real-time drug-screening platform by monitoring the inhibitory effects on enzymatic activity.
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Técnicas Biosensibles , Grafito , Acetilcolina , Compuestos de Anilina , Técnicas Biosensibles/métodos , Grafito/químicaRESUMEN
Cell spheroids are three-dimensional cell aggregates that have been widely employed in tissue engineering. Spheroid encapsulation has been explored as a method to enhance cell-cell interactions. However, the effect of hydrogel mechanical properties on spheroids, specifically soft hydrogels (<1 kPa), has not yet been studied. In this study, we determined the effect of encapsulation of stem cell spheroids by hydrogels crosslinked with different concentrations of gelatin methacryloyl (GelMA) on the functions of the stem cells. To this end, human adipose-derived stem cell (ADSC) spheroids with a defined size were prepared, and spheroid-laden hydrogels with various concentrations (5, 10, 15%) were fabricated. The apoptotic index of cells from spheroids encapsulated in the 15% hydrogel was high. The migration distance was five-fold higher in cells encapsulated in the 5% hydrogel than the 10% hydrogel. After 14 days of culture, cells from spheroids in the 5% hydrogel were observed to have spread and proliferated. Osteogenic factor and pro-angiogenic factor production in the 15% hydrogel was high. Collectively, our results indicate that the functionality of spheroids can be regulated by the mechanical properties of hydrogel, even under 1 kPa. These results indicate that spheroid-laden hydrogels are suitable for use in 3D tissue construction.
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Técnicas de Cultivo de Célula , Gelatina/química , Hidrogeles/química , Fenómenos Mecánicos , Metacrilatos/química , Esferoides Celulares , Células Madre/citología , Ingeniería de Tejidos , Apoptosis , Proliferación Celular , Supervivencia Celular , Fenómenos Químicos , Humanos , Temperatura , Ingeniería de Tejidos/métodosRESUMEN
A lateral flow assay (LFA) platform is a powerful tool for point-of-care testing (POCT), especially for self-testing. Although the LFA platform provides a simple and disposable tool for Coronavirus disease of 2019 (COVID-19) antigen (Ag) and antibody (Ab) screening tests, the lower sensitivity for low virus titers has been a bottleneck for practical applications. Herein, we report the combination of a microfluidic paper-based nanoelectrokinetic (NEK) preconcentrator and an LFA platform for enhancing the sensitivity and limit of detection (LOD). Biomarkers were electrokinetically preconcentrated onto a specific layer using the NEK preconcentrator, which was then coupled with LFA diagnostic devices for enhanced performance. Using this nanoelectrokinetic-assisted LFA (NEK-LFA) platform for self-testing, the severe acute respiratory syndrome coronavirus 2 Immunoglobulin G (SARS-CoV-2 IgG) sample was preconcentrated from serum samples. After preconcentration, the LOD of the LFA was enhanced by 32-fold, with an increase in analytical sensitivity (16.4%), which may offer a new opportunity for POCT and self-testing, especially in the COVID-19 pandemic and endemic global context.
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Técnicas Biosensibles , COVID-19 , Anticuerpos Antivirales , COVID-19/diagnóstico , Humanos , Inmunoensayo , Pandemias , SARS-CoV-2 , Sensibilidad y EspecificidadRESUMEN
The fast measurement of fibrinogen is essential in evaluating life-threatening sepsis and cardiovascular diseases. Here, we aim to utilize biomimetic plasmonic Au nanoparticles using red blood cell membranes (RBCM-AuNPs) and demonstrate nanoscale coagulation-inspired fibrinogen detection via cross-linking between RBCM-AuNPs. The proposed biomimetic RBCM-AuNPs are highly suitable for fibrinogen detection because hemagglutination, occurring in the presence of fibrinogen, induces a shift in the localized surface plasmon resonance of the NPs. Specifically, when the two ends of the fibrinogen protein are bound to receptors on separate RBCM-AuNPs, cross-linking of the RBCM-AuNPs occurs, yielding a corresponding plasmon shift within 10 min. This coagulation-inspired fibrinogen detection method, with a low sample volume, high selectivity, and high speed, could facilitate the diagnosis of sepsis and cardiovascular diseases.
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Oro , Nanopartículas del Metal , Membrana Eritrocítica , Fibrinógeno , Resonancia por Plasmón de SuperficieRESUMEN
The generation of toxic amyloid ß (Aß) oligomers is a central feature of the onset and progression of Alzheimer's disease (AD). Drug discoveries for Aß oligomer degradation have been hampered by the difficulty of Aß oligomer purification and a lack of screening tools. Here, we report a plasmonic nanoparticle amyloid corona (PNAC) for quantifying the efficacy of Aß oligomeric aggregate-degrading drugs. Our strategy is to monitor the drug-induced degradation of oligomeric aggregates by analyzing the colorimetric responses of PNACs. To test our strategy, we use Aß-degrading proteases (protease XIV and MMP-9) and subsequently various small-molecule substances that have shown benefits in the treatment of AD. We demonstrate that this strategy with PNAC can identify effective drugs for eliminating oligomeric aggregates. Thus, this approach presents an appealing opportunity to reduce attrition problems in drug discovery for AD treatment.