Cell line construction and maintenance for Lyso-IP and Endo-IP analysis of amyloid precursor protein processing v1

Lyso-IP is a method that allows for the isolation of lysosomes for proteomics and metabolomics (dx.doi.org/10.17504/protocols.io.bybjpskn; dx.doi.org/10.17504/protocols.io.bx9hpr36). We have developed an analogous approach for purification of early/sorting endosomes (Endo-IP). In addition, we have found that endolysosomal purification via Lyso-IP and Endo-IP can be coupled with a quantitative proteomics workflow to obtain snapshots of Amyloid Precursor Protein (APP) processing to its Aβ products (Park et al. in submission). Here, we describe methods for cell line construction and maintenance of 293 cells with TMEM192-3xHA and 3xFLAG-EEA1, which are used for lysosome and endosome purification, respectively, with the addition of patient mutations to APP promotes processing. Cells with endogenously tagged TMEM192 and stably expressing FLAG-EEA1 are referred to as 293EL cells, for Endo-IP and Lyso-IP. These cells were also prepared in a form that has a deletion of the APP gene (293EL;APP-/-) and the same cells reconstituted with a lentivirus stably expressing APPSw;T700N to allow functional analysis of APP processing.

Transmission Network Expansion Planning Considering Wind Power and Load Uncertainties Based on Multi-Agent DDQN

This paper presents a multi-agent Double Deep Q Network (DDQN) based on deep reinforcement learning for solving the transmission network expansion planning (TNEP) of a high-penetration renewable energy source (RES) system considering uncertainty. First, a K-means algorithm that enhances the extraction quality of variable wind and load power uncertain characteristics is proposed. Its clustering objective function considers the cumulation and change rate of operation data. Then, based on the typical scenarios, we build a bi-level TNEP model that includes comprehensive cost, electrical betweenness, wind curtailment and load shedding to evaluate the stability and economy of the network. Finally, we propose a multi-agent DDQN that predicts the construction value of each line through interaction with the TNEP model, and then optimizes the line construction sequence. This training mechanism is more traceable and interpretable than the heuristic-based methods. Simultaneously, the experience reuse characteristic of multi-agent DDQN can be implemented in multi-scenario TNEP tasks without repeated training. Simulation results obtained in the modified IEEE 24-bus system and New England 39-bus system verify the effectiveness of the proposed method.

Renovation cycle diagram

Introduction. A relatively new form of transformation of the territory in the current urban development, the renovation of quarters, requires close scientific study and methodological justification. Renovated neighborhoods, as well as projects for the construction of new houses, have individual features and characteristics. The authors hypothesize that, despite the variety of characteristics of renovated neighborhoods and projects for the construction of new houses, traditional in-line construction with the allocation of characteristic stages is possible. Materials and methods. To study the actual duration of the stages of creating objects under the renovation program (which is necessary to identify objects of similar duration), a special database was formed in the Scientific and Project Center “City Development”. As of 15.05.2021, 313 objects involved in the renovation program are included in this special database. A thorough verification of the specified database was performed. As a result, 122 objects with a spread of construction duration values from 11 months to 43.2 months are stored in the verified database. In addition, the materials of the information system for ensuring urban planning activities (ISOGD) of Moscow were used. The initial materials allowed us to identify a number of characteristic stages in the renovation program for each object. Results. To analyze the design duration of construction, the set of objects is divided into 2 parts: commissioning in 2021 and commissioning in subsequent years. For each part, histograms of the distribution of the duration of construction of houses during renovation are constructed and it is proved that the law of normal distribution is observed. A schematic model of renovation processes with multiple process durations is presented. The overall duration of the design (project stage) can take 1 year, construction — 2 years, and the combined stages: preparation of documents, relocation and demolition, will approximately take 1 year. This four-year wave cycle is then repeated the required number of times. The calendar plan of renovation for three wave objects with the allocation of the leading stream is presented. The ratio of flow processes during renovation is most clearly reflected in the constructed cyclogram of a multi-rhythmic object flow modified for renovation conditions. Combined schedules of renovation of in-line construction are constructed. Conclusions. Despite the considerable variety of renovated neighborhoods and projects for the construction of new houses, the authors of the article prove that traditional in-line construction with the allocation of characteristic stages is possible. For large contracting organizations, it is realistic to form a multi-rhythmic object flow with the allocation of the construction stage as the leading flow and calculate the renovation cyclogram.

Evaluation of Risk of Building Damage due to Deep Excavations via Numerical Modelling

In the densely populated urban environment, ground movements induced by deep excavations inevitably present a risk of damage to adjacent buildings and structures. An important and necessary aspect to justify the engineering design is to evaluate the degree of risk of such damage. This paper presents a specific procedure to evaluate the engineering design by assessing the risk level of building damage based on the deformation profiles along the ground surface which are calculated via numerical modelling and verified by empirical methods. This procedure is afterwards applied to justify the engineering design of the extension line construction of a metro project between PK0 + 344.00 and PK0 + 407.00 with the cut-and-cover excavation method. The results demonstrate that the damage risk levels of all the buildings adjacent to the excavations are within category level 0 indicating that the relevant design is acceptable while considering the risk of building damage induced by deep excavations.

Analysis of Ground Settlement Caused by Double-line TBM Tunnelling under Existing Building

TBM tunnelling is less used in the subway construction in prosperous city due to the limitation of the engineering geological conditions. The studies on the influence of the TBM construction on the existing buildings are also limited. Therefore, based on the engineering case of tunnel crossing existing building in the section of Haiboqiao ~ Xiaocunzhuang station of Qingdao Metro Line 1, the numerical model that simulates the construction process of TBM tunnelling in slightly weathered granite layer is established by three-dimensional finite difference software FLAC3D to analyze the influence of TBM tunneling on ground settlement. The comparisons of ground deformations obtained from FLAC3D and field monitoring in different construction stages of double-line tunnel have been made firstly to validate the numerical model. Then the ground settlement characteristics, differential settlement and the stress distribution of the existing building and the stress of segment structure have been analyzed. Ground settlement groove along the transverse and vertical sections occurs near the building and tunnels, and the settlement becomes smaller with the farther distance from them. The settlement curve on the cross section changes dynamically and is approximately V-shaped, and its width is about 5 ~ 6 times diameter of the tunnel. For the same cross section, the range of the settlement groove after tunnelling right line increases obviously compared with that after tunnelling left line (construction first), the settlement values also increase, and the symmetrical axis of the settlement curve is shifted to the right. This paper can provide important practical reference for relative construction engineering.