Aggregate System Research Articles

Synergy of digital economy and green economy in sustainable development policy

<p>Sustainability challenges and ICT perspectives are at the heart of current thinking on global economic and social development. The current development and growth process is based on unsustainable foundations due to irresponsible resource consumption and negative environmental impacts as well as greenhouse gas emissions. People need to find ways to integrate the digital economy and the sustainability of the green economy. Therefore, this paper firstly describes the intersection of digital economy and green economy, secondly introduces the security system of digital economy and green economy, then based on this, the SURF (Speeded-up robust features) algorithm is used to locate and improve the data aggregation system of digital economy and green economy, and finally, the algorithm simulation experiment is conducted. The experimental results found that the aggregation algorithm based on digital economy and green economy has 19% higher accuracy than the traditional algorithm. At the same time, the calculation speed is increased by three to five times. The above results show that the SURF algorithm is applied to the sustainable development research of digital economy and green economy with significant effect.</p>

Matthew effect: General design strategy of ultra‐fluorogenic nanoprobes with amplified dark–bright states in aggregates

AbstractFluorescence imaging, a key technique in biological research, frequently utilizes fluorogenic probes for precise imaging in living systems. Tetrazine is an effective emission quencher in fluorogenic probe designs, which can be selectively damaged upon bioorthogonal click reactions, leading to considerable emission enhancement. Despite significant efforts to increase the emission enhancement ratio (IAC/IBC) of tetrazine‐functionalized fluorogenic probes, the influence of molecular aggregation on the emission properties has been largely overlooked in these probe designs. In this study, we reveal that an ultrahigh IAC/IBC can be realized in the aggregate system when tetrazine is paired with aggregation‐induced emission (AIE) luminogens. Tetrazine amplifies its quenching efficiency upon aggregation and drastically reduce background emissions. Subsequent click reactions damage tetrazine and trigger significant AIE, leading to considerably enhanced IAC/IBC. We further showcase the capability of these ultra‐fluorogenic systems in selective imaging of multiple organelles in living cells. We term this unique fluorogenicity of AIE luminogen‐quencher complexes with amplified dark‐bright states as “Matthew effect” in aggregate emission, potentially providing a universal approach to attain ultrahigh IAC/IBC in diverse fluorogenic systems.

Energy landscape of perylenediimide chromophoric aggregates.

Understanding the self-assembly of conjugated organic materials at the molecular level is crucial in their potential applications as active components in electronic and optoelectronic devices. The type of aggregation significantly influences the intriguing electronic and optical characteristics differing from their constituent molecules. Perylenediimides (PDIs), electron-deficient molecules exhibiting remarkable n-type semiconducting properties, are among the most explored organic fluorescent materials due to their high fluorescence efficiency, photostability, and optoelectronic properties. PDI derivatives are reported to form well-tailored supramolecular architectures: cofacial with minor slip (H-aggregates), staggered with major slip (J-aggregates), magic angle stacking (M-aggregates), rotated (X-aggregates), rotated orthogonal ((+)-aggregates), etc. H*-aggregates are defined here as an ideal case of H-aggregate with an eclipsed configuration. Although numerous reports regarding the formation and optical properties of various PDI aggregates are known, the key driving force within the PDI units guiding the self-assembly to form distinct aggregate systems remains elusive. To unravel the molecular-level mechanisms behind the self-assembly of PDI units by probing the intermolecular interactions, symmetry-adapted perturbation theory-based energy decomposition, potential energy surface scans, and non-covalent interaction index analyses were employed on PDI dimer models. Quantum theory of atoms in molecules and frontier molecular orbital analyses were implemented on the dimer models to comprehend the effect of heteroatoms and orbital interactions in stabilising the X-aggregates over the other PDI aggregate systems. Competition between the attractive and repulsive non-covalent interactions dictates a stability order of X > H > J > M > (+) > H* for the PDI aggregate system, while in the parent perylene system, the stability order was found to be X > (+) > H > M > J > H*.

Mechanical and fracture properties of slag/steel slag-based geopolymer fully recycled aggregate concrete

Blast furnace /steel slag-based geopolymer and recycled aggregate (RA) as green alternatives to cement and natural aggregate (NA), respectively, are attracting increasing attention. This study investigated the influence of four different aggregate systems (i.e., full-NA, full-recycled coarse aggregate, full-recycled fine aggregate, and full-RA) and three different replacement ratios of steel slag (i.e., 0%, 25%, and 50% of the precursor) on the mechanical properties (compressive and splitting tensile strength) and fracture properties (fracture energy and fracture toughness) of one-part slag/steel slag-based geopolymer concrete, aiming to promote its utilization in structural engineering. The experimental results indicate that substituting ordinary Portland cement with slag-based one-part geopolymer significantly enhanced the compressive and splitting tensile strength of geopolymer natural aggregate concrete (GNAC), although it leaded to a decrease in fracture properties. The incorporation of RA in GNAC resulted in deteriorated properties, with the 28-day compressive strength being reduced by up to 5%− 19.4% and fracture energy being reduced by up to 16.8%− 43.8%. Moreover, incorporating an appropriate amount of steel slag (approximately 25%) in geopolymer fully recycled aggregate concrete (GFRAC) had minimal impact on mechanical properties, while it enhanced the fracture energy values by up to approximately 16.6%. Nevertheless, a higher content of steel slag adversely affected the relevant properties due to its lower cementitious activity compared to slag. To mitigate the drawbacks associated with steel slag, less than 25% of steel slag content is recommended considering the mechanical and fracture properties of GFRAC.

Extracellular vesicle biogenesis of three-dimensional human pluripotent stem cells in a novel Vertical-Wheel bioreactor.

Extracellular vesicles (EVs) secreted by human-induced pluripotent stem cells (hiPSCs) have great potential as cell-free therapies in various diseases, including prevention of blood-brain barrier senescence and stroke. However, there are still challenges in pre-clinical and clinical use of hiPSC-EVs due to the need for large-scale production of a large quantity. Vertical-Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC-EVs using a scalable aggregate or microcarrier-based culture system under low shear stress. EV secretion by undifferentiated hiPSCs expanded as 3-D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA-seq, respectively. The in vitro functional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3-D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA-seq. The microcarrier cultures had at least 17-23 fold higher EV secretion, and EV collection in mTeSR had 2.7-3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA-seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt-related pathways). hiPSC-EVs demonstrated the ability of stimulating proliferation and M2 polarization of microglia in vitro. HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti-aging study.

Petroleum products in wastewater and their properties

Water bodies are polluted by oil and petroleum products from various sources. The primary sources are navigation, accidents on ships and coastal areas, industrial wastewater discharges, surface wastewater from settlements, and transportation crossings of water bodies. Reducing the content of oil products in water bodies is an important task of nature protection, requiring the development of new effective methods of wastewater treatment from oil products. A systematic approach is proposed to solve this problem, including analysis of conditions of wastewater formation, study of phase-disperse composition of pollutants and their physical and chemical properties, and analysis of possible water treatment methods. According to the formation conditions, oily wastewater can be divided into domestic wastewater, surface runoff, and industrial wastewater. Depending on the formation conditions, oil products in wastewater are in different phase-disperse states with different sedimentation and aggregative stability values. Separation methods are most effective for sediment-unstable petroleum product systems and are sufficiently simple and reliable. Sedimentation stable systems of petroleum products in water need to be preliminarily destabilized. This is achieved by adding alkaline acids, mineral coagulants, or organic polyelectrolytes. Wastewater characterized by aggregative stable systems of petroleum products can be treated by degradation methods such as ozonation or sorption.

A brief history of amyloid aggregation simulations

AbstractAmyloid proteins are characterized by their tendency to aggregate into amyloid fibrils, which are often associated with devastating diseases. Aggregation pathways typically involve unfolding or misfolding of monomeric proteins and formation of transient oligomers and protofibrils before the final aggregation product is formed. The conformational dynamics and polymorphic and volatile nature of these aggregation intermediates make their characterization by experimental techniques alone insufficient and also require computational approaches. Over the past 25 years, the size of simulated amyloid aggregation systems and the length of these simulations have increased significantly. These advances are discussed here. The review includes simulation approaches that model the aggregating peptides or proteins at both the all‐atom and coarse‐grained levels, use molecular dynamics simulations or Monte Carlo sampling to simulate the conformational changes, and present results for various amyloid peptides and proteins ranging from Lys‐Phe‐Phe‐Glu (KFFE) as the smallest system to as an intermediate‐sized peptide to α‐synuclein. The presentation of the history of amyloid aggregation simulations concludes with a discussion of where the future of these simulations may lie.This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics Molecular and Statistical Mechanics > Molecular Dynamics and Monte‐Carlo Methods

SFR Modeling for Hybrid Power Systems Based on Deep Transfer Learning

A deep transfer learning method is presented for establishing the aggregated system frequency response (SFR) model of wind-thermal hybrid power systems (HPSs). In order to deal with nonlinearities and non-Gaussian disturbances, the quadratic survival information potential (QSIP) of the squared identification error is employed to construct the performance index when training recurrent neural networks (RNNs). A pre-trained SFR model is then obtained by the improved RNNs using the source domain data collected from the HPS in historical scenarios. Subsequently, the maximum mean difference is utilized to test the similarity of the HPS in historical and current scenarios. After that, the pre-trained SFR model is fine-tuned by adding some nodes to the recurrent layer and a functional link to the input layer. The SFR model of the HPS operating in current scenario can then be built based on the transferred source domain pre-trained SFR model. Simulation results illustrate that the proposed data driven modelling method can obtain accurate, effective and timely SFR model for a wind-thermal HPS with different wind speeds and load disturbances.

СОЗДАНИЕ СИСТЕМЫ ДЛЯ РЕШЕНИЯ ПРОБЛЕМ НА РЫНКЕ АГРЕГАЦИИ СКИДОК

The article is devoted to the creation of an innovative tool for aggregating discounts and promotions, designed to simplify the process of finding discounts for consumers and provide companies with the ability to monitor competitors’ discounts. The purpose of creating the tool is to react quickly to market changes and increase customer loyalty by providing more attractive offers. In the process of designing the discount aggregation system, attention is paid to all aspects necessary to create an efficient and reliable tool that meets the needs of users and provides a high level of security. The objectives of the system to be developed are formed: automation of the process of collecting and processing discount data, data filtering and validation, data classification and normalisation, efficient storage and processing of user requests. Key business processes such as data collection, data filtering and validation, classification, storage, user request processing, data personalisation and provisioning, analysis and monitoring are defined. The general algorithm of the information system is developed, input and output information arrays are allocated, the organisational and logical essence of the discount aggregation system problem solving is designed.

Implementing the subsystem of research findings control in RNPLS&T’s Single Open Information Archive (SOIA)

The RNPLS&T accomplishes research aimed at improving and developing bibliographic standards and formats, classification and indexing system, frameworks and solutions for library informatization. The materials, publications and reports on the studies make the sources for building the content of RNPLS&T’s Single Open Information Archive (SOIA) [1]. The information model of the subsystem of preservation and management of the data on the research by RNPLS&T professionals was designed on the basis of the experience of using CRIS-systems in Russian academic and research organizations. The authors attempt the analytical review of CRIS-systems implementation in Russian research and analytical organizations, and describe the RNPLS&T’s CRIS-system as a part of SOIA system. They describe in detail the metadata schemes used for presenting data on research findings in RNPLS&T’s CRIS-system.The CERIF standard is based on the data model comprising the entities of “project”, “organization” and “person”, their connections and their attributes. The extended metadata schemes in XML format used for sharing information on research exchange are oai_cerif_openaire and oai_openaire, compatible with CERIF model and supporting usability of OpenAIRE profile.The SOIA metadata exchange subsystem and OAI-PMH data provider generate metadata in oai_cerif_openaire format on the basis bibliographic records in e-catalog, data on authors, organizations and projects as presented in appropriate authority files. Actualization of data on author IDs in the international and national databases (ORCID, RSCI, WoS, Publons, Scopus, ResearchGate) in the authority files is essential.The paper is prepared within the framework of the Government Order No.of August 17, 2023, 075-01235-23-01 of August 17, 2023, theme No. 1021062311369-1-1.2.1;5.8.2;5.8.3 “Information support of research by scientists and specialists on the basis of RNPLS&T’s Open Archive as the system of scientific knowledge aggregation (FNEG-2022-0003)” for 2020–2024.

Response of gerbera under soil and soil-less production systems

Response of gerbera var. Arka Nesara was studied under different soil-less production systems with the aim to improve quality, productivity and water use efficiency, in completely randomized design with eight treatments viz., deep flow technique (DFT), nutrient film technique (NFT), aggregate wick system (AWS) with pots on NFT bench, AWS with pots on ground, AWS with grow bags on ground, AWS on soil bed, aeroponics and conventional soil-based drip system, which were replicated five times. Results revealed that AWS with pots on ground recorded maximum plant height (31.66 cm), leaf length (21.31 cm), leaf breadth (9.96 cm) and stalk diameter near neck (4.75 mm), minimum stem deviation (1.27 cm) and stem deflection (2.2o), prolonged vase life (9.2 days), greater water use efficiency (92 mL/plant/day) and water saving (83.46%). Hence, cultivation of gerbera var. Arka Nesara under aggregate wick system with pots on ground could be the superior alternative for traditional soil cultivation.

A New Method for Proportioning Sustainable, Economic, and Resilient Concrete

The ordinary Portland cement (OPC) component of concrete is the highest contributor to concrete’s cost and carbon footprint. Historically, code-writing organizations have required a high volume of paste in concrete mixtures by imposing minimum limits on the OPC content for a given application. However, high paste contents can result in dimensional instability, higher costs, higher carbon footprints, and lower durability. Minimizing the OPC content in concrete can provide economic, durability, and sustainability benefits. This study hypothesizes that the amount of OPC required to achieve some required fresh and hardened characteristics is highly dependent on the aggregate characteristics, supplementary cementing material (SCM) characteristics, and proportions of these. Given this, this research proposes using the amount of voids in the aggregate system (AV), or more specifically the paste volume-to-aggregate void ratio (PV/AV); SCM reactivity; and the SCM replacement level as key parameters to proportion concrete mixtures with minimum OPC contents to meet sustainability, economic, and resilience (SER) requirements. A new mixture proportioning procedure, referred to here as the SER proportioning method, is developed in this study based on assessing AV and identifying an optimal PV/AV that satisfies the required concrete characteristics. The results show that implementing the SER mixture proportioning method and including SCMs, or more specifically off-spec fly ashes (OFAs), can lead to significant reductions in the paste content and associated reductions in the cost and embodied carbon footprint of concrete.

Promotional effect of shaped coal gangue composite phase change agents doping in asphalt on pavement properties

To address issues related to the limited improvement in the performance of phase-change material (PCMs) modified asphalt and the unclear underlying mechanisms, this study developed two structurally stable and thermally efficient coal gangue composite phase-change agents. The impact on enhancing the properties of asphalt was systematically investigated. The study revealed that when the dosage of the phase-change agent is below 20%, there was a pronounced interactive effect between the agent and asphalt. This interaction had the capability to regulate both the non-polar and polar components within the asphalt colloid system. By controlling the dosage of the PCMs agent, it was possible to enhance the high-temperature rheological properties, high-temperature shear resistance and low-temperature flexibility of the asphalt. Additionally, this control improved the adhesion and moisture sensitivity of the PCMs modified asphalt and aggregate system. The maximum temperature-regulating effect of PCMs modified asphalt could reach up to 8.3 °C, with the functional attribute strongly correlated with the low-temperature performance of the asphalt. This research contributes to the promotion and application of PCMs in road engineering.

The Role of Agricultural Projects in Building Sustainable and Resilient Maize Value Chain in Burkina Faso

Poor seed quality and climate change significantly affect the maize value chain in Burkina Faso. To address the challenges, a catalytic project titled “Strengthening resilient seed systems in the maize value chain in Burkina Faso—from research to markets” was initiated to enhance the development of a resilient maize value chain. This study aims to assess the role of the project in developing a sustainable and resilient maize value chain. In this study, we used a mixed approach in design and implementation: qualitative research using key informants’ interviews, secondary data such as baseline survey reports, and lessons learned during the seed value chain greening intervention implemented in the Hauts-Bassins and Cascades regions of Burkina Faso. We analyzed qualitative data following the Gioia method. Kabako, a drought-tolerant hybrid seed variety, doubled crop yields in demonstration plots compared to smallholder farms and regional and national averages. Extension officers and village-based advisors (VBAs) were trained on improved seeds, composting, strip cropping, intercropping, crop rotation, and water management technologies and afterward trained smallholders. The VBAs trained smallholders on proper postharvest management practices and processing. The off-takers acted as the market. However, smallholders also sold their maize products in the informal open markets. The aggregator system was the missing link in Burkina Faso’s maize value chain. There was limited involvement of women in the project. Results obtained from this study are valuable for policymakers and value chain actors in preparing policies and filling missing gaps for exploiting the potential of the maize value chain.

Developing linguistic tools of thematic search in library information systems

Within the R&D program “Information support of research by scientists and specialists on the basis of RNPLS&T Open Archive – the system of scientific knowledge aggregation”, the RNPLS&T analyzes the use of linguistic tools of thematic search in the modern library information systems and the prospects for their development. The author defines the key common characteristics of e-catalogs of the largest Russian libraries revealed at the first stage of the analysis. Based on the specified common characteristics and detailed comparison analysis, the author outlines and substantiates the vectors for enhancing search inter faces of e-catalogs. The focus is made on linguistic tools of thematic search in library information systems; the key vectors are suggested: use of thematic search at different search levels with the clear-cut level differentiation; use of combined functionality within thematic search system; implementation of classification search in all e-catalogs; hierarchical representation of classifications; use of the matching systems for classification information retrieval languages, and in the long term classification and verbal information retrieval languages, and various verbal information retrieval languages. The author formulates practical recommendations to improve thematic search in library information systems.

The use of thromboelastography and the functional tests with double local hypoxia of the upper limb to assess the risk of thromboembolism in patients undergoing surgery

The use of thromboelastography and the functional tests with double local hypoxia of the upper limb to assess the risk of thromboembolism in patients undergoing surgery

Quality By Design Pilot Analysis of FDA Regulatory Guidance Web Data Usability for Innovators in Non-Hodgkin Lymphoma: Overcoming Challenges to CAR-T Development

Background: Non-Hodgkin lymphoma (NHL) remains a challenging hematological disease due to varied presentation and etiologies. Historically, cell/gene therapy guidance from the U.S. Food and Drug Administration (FDA) have partial utility in CAR-T development owing to the unique nature of cell-based gene modification but overlap with some key regulatory tenants of broader gene therapy. Inability to identify and apply appropriate guidance poses a challenge for next generation CAR-T immuno- and targeted therapy developers and start-ups, who are situated in a uniquely dynamic regulatory compliance landscape. Methods: A pragmatic Quality by Design (QbD) approach was taken to analyzing the usability and utility of publicly available web-based regulatory guidance via ethnographic observation of an asynchronous industry focus groups ( N = 14) of quality and translational research professionals seeking NHL regulatory guidance and/or regulatory approval information relevant to development of new CAR-T therapeutics (Fig. 1, top) over the course of approximately 10 minutes per observation. Further, a database of innovator and generic approval timelines was created for the 93 currently FDA-approved therapies based on ease of finding corresponding information. Results: Within the focus group, 7% (1/14) rated their team's confidence high in differences between recent specific FDA guidance for early phase CAR-T (Mar 2022) and prior gene and cell therapy guidance agnostic to gene therapy vs CAR-T and 79% (11/14) felt additional CAR-T manufacturing change guidance was needed. QbD stepwise analysis showed experienced users ( N = 9) clicked 10±3 pages before arriving at a desired FDA web-based guidance from the relevant framework (Fig. 1, bottom); remaining inexperienced users were unable to locate the guidance within the test period, primarily due to discrepancies in identifying appropriate classification and center (i.e. FDA CDER/CDER/CDRH) to locate the target guidance. Users had more difficulty locating information on biosimilars and generic drugs, owing in part to confusion between generic terminology on drug vs biologics/vaccines web portals. Conclusions: While current guidance has improved clarity of translational development, additional clarity and ease of access is needed for guidance on manufacturing change and implementation at non-traditional GMP sites for CAR-T to further streamline and scale these therapies. Further, generics developers, in particularly in biosimilars, may face larger hurdles in locating and applying appropriate regulatory guidance using current web-based resources. The research provides a foundation for developing merging web-based aggregation systems to improve the accessibility of regulatory content, utility, and usability of publicly available regulatory information. Such systems have the potential to improve time-to-market for innovator therapeutics and cost-effective biosimilars in NHL. This research provides insight into future web systems and natural language processing (NLP) design that could utilize merging automation to make regulatory information more accessible within key areas.

Life cycle analysis of common landfill final cover systems focusing on carbon neutrality

Carbon emissions from landfill construction and management have become a global concern. Life cycle analysis (LCA) has been widely used to assess the environmental impacts of engineered infrastructures over their lifetimes. LCA has also been applied to landfill leachate and gas management, but rarely to landfill final cover systems. This paper reports the results of an LCA of the following landfill final cover systems: compacted clay cover, geomembrane cover, cover with capillary effects (CCBE), dual capillary barrier cover, three-layer landfill cover system using natural soils, three-layer cover using recycled concrete aggregate (RCA) and biochar-amended three-layer landfill cover system using RCA. The LCA assessment of landfill cover considers the cost, carbon emissions and carbon sequestration during the production, construction and operation phases. The effects of landfill cover on global warming, freshwater eutrophication, terrestrial ecotoxicity, freshwater ecotoxicity, marine ecotoxicity and fossil resource scarcity are also evaluated. In addition, the sensitivities of cost and carbon emission to the use of electric-powered machines and transportation distance are analysed. It is revealed that the three-layer cover system using RCA and biochar has the lowest unit cost and carbon emission of all of the covers, up to 88 % and 66 % lower, respectively, than those of the other six covers. In addition, this cover system has the highest carbon sequestration rate, with a value of 47.9 kg CO2/(y·m2), four times higher than that of the compacted clay cover. Finally, this sustainable cover mitigates global warming and reduces adverse environmental impacts by up to 82 %. Therefore, the biochar amended three-layer cover system using RCA without geomembrane offers the greatest economic benefits, performs effectively in terms of the pursuit of carbon neutrality and promotes sustainable development.

Federated reinforcement learning based task offloading approach for MEC-assisted WBAN-enabled IoMT

The exponential proliferation of wearable medical apparatus and healthcare information within the framework of the Internet of Medical Things (IoMT) introduces supplementary complexities pertaining to the elevated Quality of Service (QoS) of intelligent healthcare in the forthcoming 6G era. Healthcare services and applications need ultra-reliable data transfer and processing with ultra-low latency and energy usage. Wireless Body Area Network (WBAN) and Mobile Edge Computing (MEC) technologies enabled IoMT to handle large amounts of data sensing, transmission, and processing while maintaining good QoS. Traditional frame aggregation (FA) systems in WBAN, on the other hand, create an excessive number of control frames during data transmission, resulting in significant latency and energy consumption, as well as a lack of flexibility. A Federated Reinforcement Learning (FRL) based TO Approach is recommended in this research. In the beginning, different types of service-related information were separated into queues with equal QoS needs. The duration of the FA was then automatically determined by the aggregation vertex based on energy consumption, latency, and throughput using FRL. Finally, based on the existing status, the amount of tasks offloaded was determined. The simulation results demonstrate that, as compared to the baseline schemes, the suggested FRLTO efficiently reduces energy consumption and latency while enhancing throughput and total WBAN utilization. Numerical results show that the proposed scheme improves the throughput by 37.06% and reduced the energy consumption by around 69.84% and time delay by about 6.23%, as compared to the state-of-the-art existing baseline schemes.

Amyloid fibril formation kinetics of low-pH denatured bovine PI3K-SH3 monitored by three different NMR techniques.

Introduction: Misfolding of amyloidogenic proteins is a molecular hallmark of neurodegenerative diseases in humans. A detailed understanding of the underlying molecular mechanisms is mandatory for developing innovative therapeutic approaches. The bovine PI3K-SH3 domain has been a model system for aggregation and fibril formation. Methods: We monitored the fibril formation kinetics of low pH-denatured recombinantly expressed [U-13C, 15N] labeled bovine PI3K-SH3 by a combination of solution NMR, high-resolution magic angle spinning (HR-MAS) NMR and solid-state NMR spectra. Solution NMR offers the highest sensitivity and, therefore, allows for the recording of two-dimensional NMR spectra with residue-specific resolution for individual time points of the time series. However, it can only follow the decay of the aggregating monomeric species. In solution NMR, aggregation occurs under quiescent experimental conditions. Solid-state NMR has lower sensitivity and allows only for the recording of one-dimensional spectra during the time series. Conversely, solid-state NMR is the only technique to detect disappearing monomers and aggregated species in the same sample by alternatingly recoding scalar coupling and dipolar coupling (CP)-based spectra. HR-MAS NMR is used here as a hybrid method bridging solution and solid-state NMR. In solid-state NMR and HR-MAS NMR the sample is agitated due to magic angle spinning. Results: Good agreement of the decay rate constants of monomeric SH3, measured by the three different NMR methods, is observed. Moderate MAS up to 8kHz seems to influence the aggregation kinetics of seeded fibril formation only slightly. Therefore, under sufficient seeding (1% seeds used here), quiescent conditions (solution NMR), and agitated conditions deliver similar results, arguing against primary nucleation induced by MAS as a major contributor. Using solid-state NMR, we find that the amount of disappeared monomer corresponds approximately to the amount of aggregated species under the applied experimental conditions (250µM PI3K-SH3, pH 2.5, 298K, 1% seeds) and within the experimental error range. Data can be fitted by simple mono-exponential conversion kinetics, with lifetimes τ in the 14-38h range. Atomic force microscopy confirms that fibrils substantially grew in length during the aggregation experiment. This argues for fibril elongation as the dominant growth mechanism in fibril mass (followed by the CP-based solid-state NMR signal). Conclusion: We suggest a combined approach employing both solution NMR and solid-state NMR, back-to-back, on two aliquots of the same sample under seeding conditions as an additional approach to follow monomer depletion and growth of fibril mass simultaneously. Atomic force microscopy images confirm fibril elongation as a major contributor to the increase in fibril mass.