Multitemporal analysis of land subsidence induced by open-pit mining activity using improved combined scatterer interferometry with deep learning algorithm optimization.

Mine operational safety is an important aspect of maintaining the operational continuity of a mining area. In this study, we used the InSAR time series to analyze land surface changes using the ICOPS (improved combined scatterers with optimized point scatters) method. This ICOPS method combines persistent scatterers (PS) with distributed scatterers (DS) to increase surface deformation analysis's spatial coverage and quality. One of the improvements of this study is the use of machine learning in postprocessing, based on convolutional neural networks, to increase the reliability of results. This study used data from the Sentinel-1 SAR C-band satellite during the 2016-2022 observation period at the Musan mine, North Korea. In the InSAR surface deformation time analysis, the maximum average rate of land subsidence was approximately > 15.00 cm per year, with total surface deformation of 170 cm and 70 cm for the eastern dumping area and the western dumping area, respectively. Analyzing the mechanism of land surface changes also involved evaluating the geological conditions in the Musan mining area. Our research findings show that combining machine learning and statistical methods has great potential to enhance the understanding of mine surface deformation.

Extraction of Roads Using the Archimedes Tuning Process with the Quantum Dilated Convolutional Neural Network.

Road network extraction is a significant challenge in remote sensing (RS). Automated techniques for interpreting RS imagery offer a cost-effective solution for obtaining road network data quickly, surpassing traditional visual interpretation methods. However, the diverse characteristics of road networks, such as varying lengths, widths, materials, and geometries across different regions, pose a formidable obstacle for road extraction from RS imagery. The issue of road extraction can be defined as a task that involves capturing contextual and complex elements while also preserving boundary information and producing high-resolution road segmentation maps for RS data. The objective of the proposed Archimedes tuning process quantum dilated convolutional neural network for road Extraction (ATP-QDCNNRE) technology is to tackle the aforementioned issues by enhancing the efficacy of image segmentation outcomes that exploit remote sensing imagery, coupled with Archimedes optimization algorithm methods (AOA). The findings of this study demonstrate the enhanced road-extraction capabilities achieved by the ATP-QDCNNRE method when used with remote sensing imagery. The ATP-QDCNNRE method employs DL and a hyperparameter tuning process to generate high-resolution road segmentation maps. The basis of this approach lies in the QDCNN model, which incorporates quantum computing (QC) concepts and dilated convolutions to enhance the network's ability to capture both local and global contextual information. Dilated convolutions also enhance the receptive field while maintaining spatial resolution, allowing fine road features to be extracted. ATP-based hyperparameter modifications improve QDCNNRE road extraction. To evaluate the effectiveness of the ATP-QDCNNRE system, benchmark databases are used to assess its simulation results. The experimental results show that ATP-QDCNNRE performed with an intersection over union (IoU) of 75.28%, mean intersection over union (MIoU) of 95.19%, F1 of 90.85%, precision of 87.54%, and recall of 94.41% in the Massachusetts road dataset. These findings demonstrate the superior efficiency of this technique compared to more recent methods.

Yeast lunapark regulates the formation of trans-Sey1p complexes for homotypic ER membrane fusion.

The endoplasmic reticulum (ER) consists of the nuclear envelope and a connected peripheral network of tubules and interspersed sheets. The structure of ER tubules is generated and maintained by various proteins, including reticulons, DP1/Yop1p, atlastins, and lunapark. Reticulons and DP1/Yop1p stabilize the high membrane curvature of ER tubules, and atlastins mediate homotypic membrane fusion between ER tubules; however, the exact role of lunapark remains poorly characterized. Here, using isolated yeast ER microsomes and reconstituted proteoliposomes, we directly examined the function of the yeast lunapark Lnp1p for yeast atlastin Sey1p-mediated ER fusion and found that Lnp1p inhibits Sey1p-driven membrane fusion. Furthermore, by using a newly developed assay for monitoring trans-Sey1p complex assembly, a prerequisite for ER fusion, we found that assembly of trans-Sey1p complexes was increased by the deletion of LNP1 and decreased by the overexpression of Lnp1p, indicating that Lnp1p inhibits Sey1p-mediated fusion by interfering with assembly of trans-Sey1p complexes.

GEN-Click: Genetically Encodable Click Reactions for Spatially Restricted Metabolite Labeling.

Chemical reactions for the in situ modification of biomolecules within living cells are under development. Among these reactions, bio-orthogonal reactions such as click chemistry using copper(I) and Staudinger ligation are widely used for specific biomolecule tracking in live systems. However, currently available live cell copper(I)-catalyzed azide/alkyne cycloaddition reactions are not designed in a spatially resolved manner. Therefore, we developed the "GEN-Click" system, which can target the copper(I)-catalyzed azide/alkyne cycloaddition reaction catalysts proximal to the protein of interest and can be genetically expressed in a live cell. The genetically controlled, spatially restricted, metal-catalyzed biorthogonal reaction can be used for proximity biotin labeling of various azido-bearing biomolecules (e.g., protein, phospholipid, oligosaccharides) in living cell systems. Using GEN-Click, we successfully detected local metabolite-transferring events at cell-cell contact sites.

MSH2-MSH3 promotes DNA end resection during homologous recombination and blocks polymerase theta-mediated end-joining through interaction with SMARCAD1 and EXO1.

DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.

Structures of Vac8-containing protein complexes reveal the underlying mechanism by which Vac8 regulates multiple cellular processes.

Vac8, a yeast vacuolar protein with armadillo repeats, mediates various cellular processes by changing its binding partners; however, the mechanism by which Vac8 differentially regulates these processes remains poorly understood. Vac8 interacts with Nvj1 to form the nuclear-vacuole junction (NVJ) and with Atg13 to mediate cytoplasm-to-vacuole targeting (Cvt), a selective autophagy-like pathway that delivers cytoplasmic aminopeptidase I directly to the vacuole. In addition, Vac8 associates with Myo2, a yeast class V myosin, through its interaction with Vac17 for vacuolar inheritance from the mother cell to the emerging daughter cell during cell divisions. Here, we determined the X-ray crystal structure of the Vac8-Vac17 complex and found that its interaction interfaces are bipartite, unlike those of the Vac8-Nvj1 and Vac8-Atg13 complexes. When the key amino acids present in the interface between Vac8 and Vac17 were mutated, vacuole inheritance was severely impaired in vivo. Furthermore, binding of Vac17 to Vac8 prevented dimerization of Vac8, which is required for its interactions with Nvj1 and Atg13, by clamping the H1 helix to the ARM1 domain of Vac8 and thereby preventing exposure of the binding interface for Vac8 dimerization. Consistently, the binding affinity of Vac17-bound Vac8 for Nvj1 or Atg13 was markedly lower than that of free Vac8. Likewise, free Vac17 had no affinity for the Vac8-Nvj1 and Vac8-Atg13 complexes. These results provide insights into how vacuole inheritance and other Vac8-mediated processes, such as NVJ formation and Cvt, occur independently of one another.

Human atlastins are sufficient to drive the fusion of liposomes with a physiological lipid composition.

The dynamin-like GTPase atlastin is believed to be the minimal machinery required for homotypic endoplasmic reticulum (ER) membrane fusion, mainly because Drosophila atlastin is sufficient to drive liposome fusion. However, it remains unclear whether mammalian atlastins, including the three human atlastins, are sufficient to induce liposome fusion, raising doubts about their major roles in mammalian cells. Here, we show that all human atlastins are sufficient to induce fusion when reconstituted into liposomes with a lipid composition mimicking that of the ER. Although the fusogenic activity of ATL1, which is predominantly expressed in neuronal cells, was weaker than that of ATL2 or ATL3, the addition of M1-spastin, a neuron-specific factor, markedly increased ATL1-mediated liposome fusion. Although we observed efficient fusion between ER microsomes isolated from cultured, non-neuronal cells that predominantly express ATL2-1, an autoinhibited isoform of ATL2, ATL2-1 failed to support liposome fusion by itself as reported previously, indicating that cellular factors enable ATL2-1 to mediate ER fusion in vivo.

Novel Strategy To Inhibit Transthyretin Amyloidosis via the Synergetic Effect of Chemoselective Acylation and Noncovalent Inhibitor Release.

Strategies for developing targeted covalent inhibitors (TCIs), which have the advantages of a prolonged duration of action and selectivity toward a drug target, have attracted great interest in drug discovery. Herein, we report chemoselective covalent inhibitors that specifically target lysine ε-amine groups that conjugate with an endogenous protein to prevent disease-causing protein misfolding and aggregation. These TCIs are unique because the benzoyl group is preferentially conjugated to Lys15 at the top of the T4 binding site within transthyretin (TTR) while simultaneously releasing a potent noncovalent TTR kinetic stabilizer. The potency of these covalent inhibitors is superior to tafamidis, the only FDA-approved drug for the treatment of hereditary TTR amyloidosis. In addition to investigations into the covalent modification of TTR via reverse-phase high-performance liquid chromatography, direct methods are performed to confirm and visualize the presumed covalent interaction via mass spectrometry and X-ray crystallography.

Glutamine increases stability of TPH1 mRNA via p38 mitogen-activated kinase in mouse mastocytoma cells.

Expression changes for tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in serotonin synthesis, by environmental glutamine (GLN) were examined in mouse mastocytoma-derived P815-HTR cells. GLN-treated cells exhibited a robust increase in TPH1 mRNA after a 6 h exposure to GLN. 6-Diazo-5-oxo-L-norleucine (DON), a glutamine-utilizing glutaminase inhibitor, significantly inhibited the GLN-induction of TPH1 mRNA. Nuclear run-on assays and mRNA decay experiments demonstrated that the primary mechanism leading to increased TPH1 mRNA levels was not due to transcriptional changes, but rather due to increased TPH1 RNA stability induced by GLN. Treatment with GLN also led to activation of p38 MAP kinase, but not p42/44 MAPK. In addition, SB203580, a p38 MAP kinase specific inhibitor, completely abolished the GLN-mediated increase of TPH1 mRNA levels, suggesting the pathway stabilizing TPH1 mRNA might be mediated by the activated p38 MAP kinase pathway. Additionally, SB203580 significantly reduced the stability of TPH1 mRNA, and this reduction of the stability was not affected by GLN in the culture medium, implying a sequential signaling from GLN being mediated by p38 MAP kinase, resulting in alteration of TPH1 mRNA stability. TPH1 mRNA stability loss was also dependent on de novo protein synthesis as shown by treatment of cells with a transcriptional/translational blocker. We provide evidence that TPH1 mRNA levels are increased in response to increased exogenous GLN in mouse mastocytoma cells via a stabilization of TPH1 mRNA due to the activity of the p38 MAP kinase.

Structural basis for mitoguardin-2 mediated lipid transport at ER-mitochondrial membrane contact sites.

The endoplasmic reticulum (ER)-mitochondria contact site (ERMCS) is crucial for exchanging biological molecules such as phospholipids and Ca2+ ions between these organelles. Mitoguardin-2 (MIGA2), a mitochondrial outer membrane protein, forms the ERMCS in higher eukaryotic cells. Here, we report the crystal structures of the MIGA2 Lipid Droplet (LD) targeting domain and the ER membrane protein VAPB bound to the phosphorylated FFAT motif of MIGA2. These structures reveal that the MIGA2 LD targeting domain has a large internal hydrophobic pocket that accommodates phospholipids and that two phosphorylations of the FFAT motif are required for tight interaction of MIGA2 with VAPB, which enhances the rate of lipid transport. Further biochemical studies show that MIGA2 transports phospholipids between membranes with a strong preference for binding and trafficking phosphatidylserine (PS). These results provide a structural and molecular basis for understanding how MIGA2 mediates the formation of ERMCS and facilitates lipid trafficking at the ERMCS.

A chemical tool for blue light-inducible proximity photo-crosslinking in live cells.

We developed a proximity photo-crosslinking method (Spotlight) with a 4-azido-N-ethyl-1,8-naphthalimide (AzNP) moiety that can be converted to reactive aryl nitrene species using ambient blue light-emitting diode light. Using an AzNP-conjugated HaloTag ligand (VL1), blue light-induced photo-crosslinked products of various HaloTag-conjugated proteins of interest were detected in subcellular spaces in live cells. Chemical or heat stress-induced dynamic changes in the proteome were also detected, and photo-crosslinking in the mouse brain tissue was enabled. Using Spotlight, we further identified the host interactome of SARS-CoV-2 nucleocapsid (N) protein, which is essential for viral genome assembly. Mass analysis of the VL1-crosslinked product of N-HaloTag in HEK293T cells showed that RNA-binding proteins in stress granules were exclusively enriched in the cross-linked samples. These results tell that our method can reveal the interactome of protein of interest within a short distance in live cells.

Augmented ERAD (ER-associated degradation) activity in chondrocytes is necessary for cartilage development and maintenance.

Chondrocytes secrete massive extracellular matrix (ECM) molecules that are produced, folded, and modified in the endoplasmic reticulum (ER). Thus, the ER-associated degradation (ERAD) complex-which removes misfolded and unfolded proteins to maintain proteostasis in the ER- plays an indispensable role in building and maintaining cartilage. Here, we examined the necessity of the ERAD complex in chondrocytes for cartilage formation and maintenance. We show that ERAD gene expression is exponentially increased during chondrogenesis, and disruption of ERAD function causes severe chondrodysplasia in developing embryos and loss of adult articular cartilage. ERAD complex malfunction also causes abnormal accumulation of cartilage ECM molecules and subsequent chondrodysplasia. ERAD gene expression is decreased in damaged cartilage from patients with osteoarthritis (OA), and disruption of ERAD function in articular cartilage leads to cartilage destruction in a mouse OA model.

Convolutional neural network (CNN) with metaheuristic optimization algorithms for landslide susceptibility mapping in Icheon, South Korea.

Landslides are a geological hazard that can pose a serious threat to human health and the environment of highlands or mountain slopes. Landslide susceptibility mapping is an essential tool for predicting and mitigating landslides. This study aimed to investigate the application of deep learning algorithms based on convolutional neural networks (CNNs) with metaheuristic optimization algorithms, namely the grey wolf optimizer (GWO) and imperialist competitive algorithm (ICA), to landslide susceptibility mapping. The study area was Icheon City, South Korea, for which an accurate landslide inventory dataset was available. The landslide inventory map was prepared and randomly divided into datasets of 70% for training and 30% for validation. Additionally, 18 landslide-related factors, including geo-environmental and topo-hydrological factors, were considered as predictive variables. The models were compared using area under the curve (AUC) values in receiver operating characteristic (ROC) curve analysis. The validation results showed that optimized models based on CNN-GWO (AUC = 0.876, RMSE = 0.08) and CNN-ICA (AUC = 0.852, RMSE = 0.09) outperformed the standalone CNN model (AUC = 0.847, RMSE = 0.12). Nevertheless, the CNN model outperformed previous research that used a machine learning algorithm alone. Thus, the deep learning algorithm with optimization algorithms proposed in this study can generate more suitable models for landslide susceptibility mapping in the study area due to its improved accuracy.

Mitoquinone Inactivates Mitochondrial Chaperone TRAP1 by Blocking the Client Binding Site.

Heat shock protein 90 (Hsp90) family proteins are molecular chaperones that modulate the functions of various substrate proteins (clients) implicated in pro-tumorigenic pathways. In this study, the mitochondria-targeted antioxidant mitoquinone (MitoQ) was identified as a potent inhibitor of mitochondrial Hsp90, known as a tumor necrosis factor receptor-associated protein 1 (TRAP1). Structural analyses revealed an asymmetric bipartite interaction between MitoQ and the previously unrecognized drug binding sites located in the middle domain of TRAP1, believed to be a client binding region. MitoQ effectively competed with TRAP1 clients, and MitoQ treatment facilitated the identification of 103 TRAP1-interacting mitochondrial proteins in cancer cells. MitoQ and its redox-crippled SB-U014/SB-U015 exhibited more potent anticancer activity in vitro and in vivo than previously reported mitochondria-targeted TRAP1 inhibitors. The findings indicate that targeting the client binding site of Hsp90 family proteins offers a novel strategy for the development of potent anticancer drugs.

Design and Synthesis of TRAP1 Selective Inhibitors: H-Bonding with Asn171 Residue in TRAP1 Increases Paralog Selectivity.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is overexpressed in the mitochondria of various cancer cells, reprograms cellular metabolism to enable cancer cells to adapt to harsh tumor environments. As inactivation of TRAP1 induces massive apoptosis in cancer cells in vitro and in vivo, the development of TRAP1-selective inhibitors has become an attractive approach. A series of purine-8-one and pyrrolo[2,3-d]pyrimidine derivatives was developed based on TRAP1 structure and identified to be highly selective in vitro for TRAP1 over the paralogous enzymes, Hsp90α and Grp94. The TRAP1-selective inhibition strategy via utilization of the Asn171 residue of the ATP-lid was investigated using X-ray crystallography and molecular dynamics simulation studies. Among various synthesized potent TRAP1 inhibitors, 5f possessed a 65-fold selectivity over Hsp90α and a 13-fold selectivity over Grp94. Additionally, 6f had a half-maximal inhibitory concentration (IC50) of 63.5 nM for TRAP1, with a 78-fold and 30-fold selectivity over Hsp90α and Grp94, respectively.

Development of pyrazolo[3,4-d]pyrimidine-6-amine-based TRAP1 inhibitors that demonstrate in vivo anticancer activity in mouse xenograft models.

TNF Receptor Associated Protein 1 (TRAP1) is a mitochondrial paralog of Hsp90 related to the promotion of tumorigenesis in various cancers via maintaining mitochondrial integrity, reducing the production of reactive oxygen species, and reprogramming cellular metabolism. Consequently, Hsp90 and TRAP1 have been targeted to develop cancer therapeutics. Herein, we report a series of pyrazolo[3,4-d]pyrimidine derivatives that are mitochondria-permeable TRAP1 inhibitors. Structure-based drug design guided the optimization of potency, leading to the identification of compounds 47 and 48 as potent TRAP1 and Hsp90 inhibitors with good metabolic and plasma stability as well as acceptable CYP and hERG inhibition. X-ray co-crystallization studies confirmed both 47 and 48 interact with the ATP binding pocket in the TRAP1 protein. Compounds 47 and 48 demonstrated excellent anticancer efficiency in various cancer cells, with limited toxicity over normal hepatocyte and prostate cells. Mouse PC3 xenograft studies showed 47 and 48 significantly reduced tumor growth.

γ-Secretase Partitioning into Lipid Bilayers Remodels Membrane Microdomains after Direct Insertion.

γ-Secretase is a multisubunit complex that catalyzes intramembranous cleavage of transmembrane proteins. The lipid environment forms membrane microdomains that serve as spatio-temporal platforms for proteins to function properly. Despite substantial advances in the regulation of γ-secretase, the effect of the local membrane lipid microenvironment on the regulation of γ-secretase is poorly understood. Here, we characterized and quantified the partitioning of γ-secretase and its substrates, the amyloid precursor protein (APP) and Notch, into lipid bilayers using solid-supported model membranes. Notch substrate is preferentially localized in the liquid-disordered (Ld) lipid domains, whereas APP and γ-secretase partition as single or higher complex in both phases but highly favor the ordered phase, especially after recruiting lipids from the ordered phase, indicating that the activity and specificity of γ-secretase against these two substrates are modulated by membrane lateral organization. Moreover, time-elapse measurements reveal that γ-secretase can recruit specific membrane components from the cholesterol-rich Lo phase and thus creates a favorable lipid environment for substrate recognition and therefore activity. This work offers insight into how γ-secretase and lipid modulate each other and control its activity and specificity.

Impaired DNA-binding affinity of novel PAX6 mutations.

Mutations in human PAX6 gene are associated with various congenital eye malformations including aniridia, foveal hypoplasia, and congenital nystagmus. These various phenotypes may depend on the mutation spectrums that can affect DNA-binding affinity, although this hypothesis is debatable. We screened PAX6 mutations in two unrelated patients with congenital nystagmus, and measured DNA-binding affinity through isothermal titration calorimetry (ITC). To elucidate phenotypic differences according to DNA-binding affinity, we also compared DNA-binding affinity among the previously reported PAX6 missense mutations within the linker region between two subdomains of the paired domain (PD). We identified two novel mutations of PAX6 gene: c.214 G > T (p.Gly72Cys) and c.249_250delinsCGC (p.Val84Alafs*8). Both were located within the linker region between the two subdomains of the PD. ITC measurement revealed that the mutation p.Val84Alafs*8 had no DNA-binding affinity, while the p.Gly72Cys mutation showed a decreased binding affinity (Kd = 0.58 µM) by approximately 1.4 times compared to the wild type-PAX6 (Kd = 0.41 µM). We also found that there was no close relationship between DNA-binding affinity and phenotypic differences. Our results suggest that the DNA-binding affinity alone might be insufficient to determine PAX6-related phenotypes, and that other modifier genes or environmental factors might affect phenotypes of the PAX6 gene.

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy.

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging.