Integral membrane proteins isolated from cellular environment often lose activity and native conformation required for functional analyses and structural studies. Even in their native state, they ...lack sufficient surfaces to form crystal contacts. Furthermore, most of them are too small for cryogenic electron microscopy detection and too big for solution NMR. To overcome these difficulties, we recently developed a strategy to stabilize the folded state of membrane proteins by restraining their two termini with a self-assembling protein coupler. The termini-restrained membrane proteins from distinct functional families retain their activities and show increased stability and yield. This strategy enables their structure determination at near-atomic resolution by facilitating the entire pipeline from crystallization, crystal identification, diffraction enhancement and phase determination, to electron density improvement. Furthermore, stabilization of membrane proteins enables their biochemical and biophysical characterization. Here we present the protocol of membrane protein engineering (2 weeks), quality assessment (1-2 weeks), protein production (1-6 weeks), crystallization (1-2 weeks), diffraction improvement (1-3 months) and crystallographic data analysis (1 week). This protocol is intended not only for structural biologists, but also for biochemists, biophysicists and pharmaceutical scientists whose research focuses on membrane proteins.
In cognitive radios, wideband sequential sensing plays an important role, which is able to quickly identify temporary available transmission opportunities by adaptively allocating sensing resources. ...This paper proposes a Markov decision process for modelling the optimal control of sequential sensing, which provides a general formulation capturing various practical features, including sampling cost, sensing requirement, sensing budget etc. For solving the optimal sensing policy, a model‐augmented deep reinforcement learning algorithm is proposed, which enjoys high learning stability and efficiency, compared to conventional reinforcement learning algorithms.
This paper proposes a Markov decision process for modelling the optimal control of sequential sensing, which provides a general formulation capturing various practical features, including sampling cost, sensing requirement, sensing budget etc. For solving the optimal sensing policy, a model‐augmented deep reinforcement learning algorithm is proposed, which enjoys high learning stability and efficiency, compared to conventional reinforcement learning algorithms.
Crystal structures of membrane proteins are highly desired for their use in the mechanistic understanding of their functions and the designing of new drugs. However, obtaining the membrane protein ...structures is difficult. One way to overcome this challenge is with protein fusion methods, which have been successfully used to determine the structures of many membrane proteins, including receptors, enzymes and adhesion molecules. Existing fusion strategies can be categorized into the N or C terminal fusion, the insertion fusion and the termini restraining. The fusions facilitate protein expression, purification, crystallization and phase determination. Successful applications often require further optimization of protein fusion linkers and interactions, whose design can be facilitated by a shared helix strategy and by AlphaFold prediction in the future.
Dissolved organic nitrogen (DON) plays a crucial role in biogeochemical processes of nitrogen (N) and has become a non‐negligible source of increasing groundwater nitrogen contamination. Therefore, ...the extraction accuracy of DON in unsaturated zones is essential for sound understanding of N transport, transformation behaviour and contamination processes. However, there is no uniform extraction standard for measuring DON content in the unsaturated zone. Moreover, the influences of various extraction conditions on the DON measurement are not clear. Hereby, we are the first to address the issues associated with all the extraction indicators (extractant types and concentrations, extraction duration, frequency, and solution volume‐to‐soil weight ratio) for the DON extraction accuracy in unsaturated zones, and present a comprehensive influence analyses. Our results revealed that extraction conditions could significantly influence the extraction accuracy of DON and that the principal impact factor was the concentration of the extractant. We also found that the widely used previous extraction approaches (high‐salinity extraction method) would induce high uncertainty in measuring the DON content in unsaturated zones of different areas. Furthermore, the opposing influences from the ions of extractants can seriously affect the assessment of DON content and hinder comparability even using the same extraction method, which could severely affect the assessment of the DON content in unsaturated zones. Such uncertainty in measurements will eventually influence the observation and evaluation of groundwater nitrogen contamination. Our results demonstrated that the use of the former DON extraction method without considering the research purposes and the specific study areas would lead to a strong bias. Based on detailed response surface analysis, we provide a comprehensive analysis related to the accuracy of DON extraction, which is conducive to improving the extraction accuracy of nitrogen research in global groundwater hydrological processes in the future.
A comprehensive analysis of DON extraction accuracy was firstly presented. Opposite effects from ions of extractants can seriously affect DON accuracy.
Abstract
Thermoelectrics enable direct heat-to-electricity transformation, but their performance has so far been restricted by the closely coupled carrier and phonon transport. Here, we demonstrate ...that the quantum gaps, a class of planar defects characterized by nano-sized potential wells, can decouple carrier and phonon transport by selectively scattering phonons while allowing carriers to pass effectively. We choose the van der Waals gap in GeTe-based materials as a representative example of the quantum gap to illustrate the decoupling mechanism. The nano-sized potential well of the quantum gap in GeTe-based materials is directly visualized by in situ electron holography. Moreover, a more diffused distribution of quantum gaps results in further reduction of lattice thermal conductivity, which leads to a peak
ZT
of 2.6 at 673 K and an average ZT of 1.6 (323–723 K) in a GeTe system. The quantum gap can also be engineered into other thermoelectrics, which provides a general method for boosting their thermoelectric performance.
Animal cells within a tissue typically display a striking regularity in their size. To date, the molecular mechanisms that control this uniformity are still unknown. We have previously shown that ...size uniformity in animal cells is promoted, in part, by size-dependent regulation of G1 length. To identify the molecular mechanisms underlying this process, we performed a large-scale small molecule screen and found that the p38 MAPK pathway is involved in coordinating cell size and cell cycle progression. Small cells display higher p38 activity and spend more time in G1 than larger cells. Inhibition of p38 MAPK leads to loss of the compensatory G1 length extension in small cells, resulting in faster proliferation, smaller cell size and increased size heterogeneity. We propose a model wherein the p38 pathway responds to changes in cell size and regulates G1 exit accordingly, to increase cell size uniformity.
The intramembrane vitamin K epoxide reductase (VKOR) supports blood coagulation in humans and is the target of the anticoagulant warfarin. VKOR and its homologues generate disulphide bonds in ...organisms ranging from bacteria to humans. Here, to better understand the mechanism of VKOR catalysis, we report two crystal structures of a bacterial VKOR captured in different reaction states. These structures reveal a short helix at the hydrophobic active site of VKOR that alters between wound and unwound conformations. Motions of this 'horizontal helix' promote electron transfer by regulating the positions of two cysteines in an adjacent loop. Winding of the helix separates these 'loop cysteines' to prevent backward electron flow. Despite these motions, hydrophobicity at the active site is maintained to facilitate VKOR catalysis. Biochemical experiments suggest that several warfarin-resistant mutations act by changing the conformation of the horizontal helix. Taken together, these studies provide a comprehensive understanding of VKOR function.
Abstract
Osteoporosis is a common bone disease characterized by loss of bone mass, reduced bone strength, and deterioration of bone microstructure. ROS-induced oxidative stress plays an important ...role in osteoporosis. However, the biomarkers and molecular mechanisms of oxidative stress are still unclear. We obtained the datasets from the Gene Expression Omnibus (GEO) database, and performed differential analysis, Venn analysis, and weighted correlation network analysis (WGCNA) analysis out the hub genes. Then, the correlation between inflammatory factors and hub genes was analyzed, and a Mendelian randomization (MR) analysis was performed on cytokines and osteoporosis outcomes. In addition, “CIBERSORT” was used to analyze the infiltration of immune cells and single-cell RNA-seq data was used to analyze the expression distribution of hub genes and cell–cell communications. Finally, we collected human blood samples for RT-qPCR and Elisa experiments, the miRNA-mRNA network was constructed using the miRBase database, the 3D structure was predicted using the RNAfold, Vfold3D database, and the drug sensitivity analysis was performed using the RNAactDrug database. We obtained three differentially expressed genes associated with oxidative stress: DBH, TAF15, and STAT4 by differential, WGCNA clustering, and Venn screening analyses, and further analyzed the correlation of these 3 genes with inflammatory factors and immune cell infiltration and found that STAT4 was significantly and positively correlated with IL-2. Single-cell data analysis showed that the STAT4 gene was highly expressed mainly in dendritic cells and monocytes. In addition, the results of RT-qPCR and Elisa experiments verified that the expression of STAT4 was consistent with the previous analysis, and a significant causal relationship between IL-2 and STAT4 SNPs and osteoporosis was found by Mendelian randomization. Finally, through miRNA-mRNA network and drug sensitivity analysis, we analyzed to get Palbociclib/miR-141-3p/STAT4 axis, which can be used for the prevention and treatment of osteoporosis. In this study, we proposed the Palbociclib/miR-141-3p/STAT4 axis for the first time and provided new insights into the mechanism of oxidative stress in osteoporosis.
Background:
Recent studies have demonstrated the significance of the
DEAD-box helicase 5
(
DDX5
) gene, which is involved in pathways concerning the modification of RNA structures.
DDX5
functions as ...a coregulator of cellular transcription and splicing, and participates in the processing of small noncoding RNAs. The aberrant regulation of
DDX5
expression possibly plays a significant role in the genesis of cancer. However, there are no comprehensive pan-cancer studies on
DDX5
. This study is the first to conduct a pan-cancer analysis of
DDX5
for aiding the diagnosis and treatment of cancer.
Methods:
The gene expression, genetic alterations, protein phosphorylation, promoter methylation, immune infiltration, and enrichment analyses of
DDX5
were performed using data retrieved from The Cancer Genome Atlas (TCGA), Genotype-tissue Expression (GTEx), Human Protein Atlas (HPA), Tumor Immunological Estimation Resource 2.0 (TIMER2.0), Gene Expression Profiling Interactive Analysis (GEPIA), DNA methylation interactive visualization database (DNMIVD), and Search Tool for the Retrieval of Interaction Genes/Proteins (STRING). Data analyses were performed with the R software and other webtools.
Results:
The expression of DDX5 mRNA decreased significantly in 17 cancer types, but increased significantly in eight cancer types. The enhanced expression of DDX5 mRNA in the tumor samples was related to decreased overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS) in three cancers, but increased OS, PFI, and DSS in other cancers. The DNA promoter methylation level was significantly reduced in eight cancer types, and there were exceptions in the methylation levels of the
DDX5
promoter in four cancer types. The expression of DDX5 mRNA was highly correlated with the infiltration of CD8
+
T cells, cancer-associated fibroblasts, and B cells in a wide variety of malignancies. The findings revealed a strong association between
DDX5
and its co-expressed genes in numerous cancer types. Enrichment analysis suggested that
DDX5
was associated with multiple cellular pathways, including RNA splicing, Notch signaling pathway, and viral carcinogenesis, which was consistent with the results of previous studies.
Conclusion:
The findings obtained herein provide further information on the oncogenic potential of
DDX5
in diverse tumor types. We propose that
DDX5
has important roles in tumor immunity and the diagnosis of cancer.
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