Correlative montage parallel array cryo-tomography for in situ structural cell biology.

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here we present robust tools for montage parallel array cryo-tomography (MPACT) tailored for vitrified specimens. The combination of correlative cryo-fluorescence microscopy, focused-ion-beam milling, substrate micropatterning, and MPACT supports studies that contextually define the three-dimensional architecture of cells. To further extend the flexibility of MPACT, tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

Cryo-electron Microscopic Analysis of Single-Pass Transmembrane Receptors.

Single-pass transmembrane receptors (SPTMRs) represent a diverse group of integral membrane proteins that are involved in many essential cellular processes, including signal transduction, cell adhesion, and transmembrane transport of materials. Dysregulation of the SPTMRs is linked with many human diseases. Despite extensive efforts in past decades, the mechanisms of action of the SPTMRs remain incompletely understood. One major hurdle is the lack of structures of the full-length SPTMRs in different functional states. Such structural information is difficult to obtain by traditional structural biology methods such as X-ray crystallography and nuclear magnetic resonance (NMR). The recent rapid development of single-particle cryo-electron microscopy (cryo-EM) has led to an exponential surge in the number of high-resolution structures of integral membrane proteins, including SPTMRs. Cryo-EM structures of SPTMRs solved in the past few years have tremendously improved our understanding of how SPTMRs function. In this review, we will highlight these progresses in the structural studies of SPTMRs by single-particle cryo-EM, analyze important structural details of each protein involved, and discuss their implications on the underlying mechanisms. Finally, we also briefly discuss remaining challenges and exciting opportunities in the field.

New Insights into MeHg Accumulation in Rice (Oryza sativa L.): Evidence from Cysteine.

The intake of methylmercury (MeHg)-contaminated rice poses immense health risks to rice consumers. However, the mechanisms of MeHg accumulation in rice plants are not entirely understood. The knowledge that the MeHg-Cysteine complex was dominant in polished rice proposed a hypothesis of co-transportation of MeHg and cysteine inside rice plants. This study was therefore designed to explore the MeHg accumulation processes in rice plants by investigating biogeochemical associations between MeHg and amino acids. Rice plants and underlying soils were collected from different Hg-contaminated sites in the Wanshan Hg mining area. The concentrations of both MeHg and cysteine in polished rice were higher than those in other rice tissues. A significant positive correlation between MeHg and cysteine in rice plants was found, especially in polished rice, indicating a close geochemical association between cysteine and MeHg. The translocation factor (TF) of cysteine showed behavior similar to that of the TF of MeHg, demonstrating that these two chemical species might share a similar transportation mechanism in rice plants. The accumulation of MeHg in rice plants may vary due to differences in the molar ratios of MeHg to cysteine and the presence of specific amino acid transporters. Our results suggest that cysteine plays a vital role in MeHg accumulation and transportation inside rice plants.

G4LDB 2.2: a database for discovering and studying G-quadruplex and i-Motif ligands.

Noncanonical nucleic acid structures, such as G-quadruplex (G4) and i-Motif (iM), have attracted increasing research interests because of their unique structural and binding properties, as well as their important biological activities. To date, thousands of small molecules that bind to varying G4/iM structures have been designed, synthesized and tested for diverse chemical and biological uses. Because of the huge potential and increasing research interests on G4-targeting ligands, we launched the first G4 ligand database G4LDB in 2013. Here, we report a new version, termed G4LDB 2.2 (http://www.g4ldb.com), with upgrades in both content and function. Currently, G4LDB2.2 contains >3200 G4/iM ligands, ∼28 500 activity entries and 79 G4-ligand docking models. In addition to G4 ligand library, we have also added a brand new iM ligand library to G4LDB 2.2, providing a comprehensive view of quadruplex nucleic acids. To further enhance user experience, we have also redesigned the user interface and optimized the database structure and retrieval mechanism. With these improvements, we anticipate that G4LDB 2.2 will serve as a comprehensive resource and useful research toolkit for researchers across wide scientific communities and accelerate discovering and validating better binders and drug candidates.

Amine Switchable Hydrophilic Solvent Vortex-Assisted Homogeneous Liquid-Liquid Microextraction and GC-MS for the Enrichment and Determination of 2, 6-DIPA Additive in Biodegradable Film.

2, 6-diisopropylaniline (2, 6-DIPA) is a crucial non-intentionally organic additive that allows the assessment of the production processes, formulation qualities, and performance variations in biodegradable mulching film. Moreover, its release into the environment may have certain effects on human health. Hence, this study developed simultaneous heating hydrolysis-extraction and amine switchable hydrophilic solvent vortex-assisted homogeneous liquid-liquid microextraction for the gas chromatography-mass spectrometry analysis of the 2, 6-DIPA additive and its corresponding isocyanates in poly(butylene adipate-co-terephthalate) (PBAT) biodegradable agricultural mulching films. The heating hydrolysis-extraction conditions and factors influencing the efficiency of homogeneous liquid-liquid microextraction, such as the type and volume of amine, homogeneous-phase and phase separation transition pH, and extraction time were investigated and optimized. The optimum heating hydrolysis-extraction conditions were found to be a H2SO4 concentration of 2.5 M, heating temperature of 87.8 °C, and hydrolysis-extraction time of 3.0 h. As a switchable hydrophilic solvent, dipropylamine does not require a dispersant. Vortex assistance is helpful to speed up the extraction. Under the optimum experimental conditions, this method exhibits a better linearity (0.0144~7.200 µg mL-1 with R = 0.9986), low limit of detection and quantification (0.0033 µg g-1 and 0.0103 µg g-1), high extraction recovery (92.5~105.4%), desirable intra- and inter-day precision (relative standard deviation less than 4.1% and 4.7%), and high enrichment factor (90.9). Finally, this method was successfully applied to detect the content of the additive 2, 6-DIPA in PBAT biodegradable agricultural mulching films, thus facilitating production process monitoring or safety assessments.

Structural organization of the C1b projection within the ciliary central apparatus.

Motile cilia have a '9+2' structure containing nine doublet microtubules and a central apparatus (CA) composed of two singlet microtubules with associated projections. The CA plays crucial roles in regulating ciliary motility. Defects in CA assembly or function usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of most CA projections remain largely unknown. Here, we combined genetic, proteomic and cryo-electron tomographic approaches to compare the CA of wild-type Chlamydomonas reinhardtii with those of three CA mutants. Our results show that two proteins, FAP42 and FAP246, are localized to the L-shaped C1b projection of the CA, where they interact with the candidate CA protein FAP413. FAP42 is a large protein that forms the peripheral 'beam' of the C1b projection, and the FAP246-FAP413 subcomplex serves as the 'bracket' between the beam (FAP42) and the C1b 'pillar' that attaches the projection to the C1 microtubule. The FAP246-FAP413-FAP42 complex is essential for stable assembly of the C1b, C1f and C2b projections, and loss of these proteins leads to ciliary motility defects.

Nitrogen application and differences in leaf number retained after topping affect the tobacco (Nicotiana tabacum) transcriptome and metabolome.

BACKGROUND: Agronomic treatments such as the application of nitrogen fertilizer and topping (removal of the inflorescence and top leaves) cause substantial changes in plant metabolism. To explore these changes, we conducted comparative transcriptomic and metabolomic analyses of leaves collected from four positions along the stem on plants exposed to two nitrogen doses and with different numbers of leaves retained after topping in tobacco (Nicotiana tabacum). RESULTS: We identified 13,330 unique differentially expressed genes and 32 differentially abundant metabolites. Through RNA-seq and WGCNA analyze, we constructed 2 co-expression networks (green and blue) highly correlation to N application and leaf number retained, predicted a hub gene NtGER3 may play an important role in N metabolism related to amino acid (cysteine) through CK pathway in tobacco leaves, NtARFs may participated in modulating the auxin signal and N in bottom leaves and NtRAP2.12 as key gene involved in N regulation by ethylene pathway. What's more, our data prove C/N transformation and balance affect the "source - flow - sink" redistribution and remobilization in tobacco during growth and development process. CONCLUSIONS: Overall, this comparative transcriptomics study provides novel insight into the complex molecular mechanisms underlying plant responses to different levels of nitrogen application and the number of leaves remaining after topping in plants.

Anthocyanin improves kidney function in diabetic kidney disease by regulating amino acid metabolism.

BACKGROUND: Diabetic kidney disease (DKD) is among the most important causes for chronic kidney disease. Anthocyanins (ANT) are polyphenolic compounds present in various food and play an important role in ameliorating hyperglycemia and insulin sensitivity. However, the effects of ANT in DKD are still poorly understood. This study aimed to investigate the effect of ANT (cyanidin-3-O-glucoside [C3G]) on the renal function of DKD, and whether the anti-DKD effect of ANT is related to metabolic pathways. METHODS: To explore the role of ANT in DKD, we performed the examination of blood glucose, renal function, and histopathology. As for the mechanism, we designed the label-free quantification proteomics and nontargeted metabolomics analysis for kidney and serum. Subsequently, we revealed the anti-DKD effect of ANT through the bioinformatic analysis. RESULTS: We showed that the fasting blood glucose level (- 6.1 mmol/L, P = 0.037), perimeter of glomerular lesions (- 24.1 µm, P = 0.030), fibrosis score of glomerular (- 8.8%, P = 0.002), and kidney function (Cystatin C: - 701.4 pg/mL, P = 0.043; urine creatinine: - 701.4 mmol/L, P = 0.032) were significantly alleviated in DKD mice after ANT treatment compared to untreated in the 20th week. Further, proteins and metabolites in the kidneys of DKD mice were observed to be dramatically altered due to changes in amino acid metabolism with ANT treatment; mainly, taurine and hypotaurine metabolism pathway was upregulated (P = 0.0001, t value = 5.97). Furthermore, upregulated tryptophan metabolism (P < 0.0001, t value = 5.94) and tyrosine metabolism (P = 0.0037, t value = 2.91) pathways had effects on serum of DKD mice as responsed ANT regulating. CONCLUSIONS: Our results suggested that prevention of the progression of DKD by ANT could be related to the regulation of amino acid metabolism. The use of dietary ANT may be one of the dietary strategies to prevent and treat DKD.

LncRNA HOXA-AS2 facilitates prostate cancer progression by inhibiting miR-885-5p to upregulate KDM5B.

BACKGROUND: The regulatory network of competing endogenous RNAs (ceRNAs) affects tumorigenesis. In this study, we aimed to investigate the mechanism by which long non-coding RNA (lncRNA) HOXA-AS2 promotes prostate cancer (PCa) progression. METHODS: The expression levels of HOXA-AS2, miR-885-5p, and KDM5B in PCa tissues and cell lines were evaluated by qRT-PCR or western blotting. CCK-8 assay, caspase-3 activity assay, flow cytometry, and scratch test revealed changes in cell proliferation, caspase-3 activity, apoptosis, and migration, respectively. Luciferase and radioimmunoprecipitation assays were used to evaluate the correlation among HOXA-AS2, miR-885-5p, and KDM5B expression profiles. RESULTS: HOXA-AS2 expression level was elevated in PCa tissues and cells. Silencing of HOXA-AS2 suppressed proliferation and migration and facilitated apoptosis in PCa cells. HOXA-AS2 competitively adsorbed miR-885-5p, thereby blocking the effect of HOXA-AS2 knockdown by the miR-885-5p inhibitor in PCa cells. Moreover, KDM5B, a target of miR-885-5p, neutralized the function of miR-885-5p in PCa cells. CONCLUSIONS: This study revealed a potential ceRNA regulatory pathway in which HOXA-AS2 affects KDM5B expression levels by sponging miR-885-5p to promote PCa development and progression.

Determination of Residual Diisocyanates and Related Diamines in Biodegradable Mulch Films Using N-Ethoxycarbonylation Derivatization and GC-MS.

Diisocyanates are highly reactive compounds with two functional isocyanate groups. The exposure of diisocyanates is associated with severely adverse health effects, such as asthma, inflammation in the respiratory tract, and cancer. The hydrolysis product from diisocyanates to related diamines is also a potential carcinogen. Here, we developed an effective, accurate, and precise method for simultaneous determination of residual diisocyanates and related diamines in biodegradable mulch films, based on N-ethoxycarbonylation derivatization and gas chromatography-mass spectrometry. The method development included the optimization of ultrasonic hydrolysis and extraction, screening of N-ethoxycarbonylation conditions with ethyl chloroformate, evaluation of the diamines degradation, and analysis of the fragmentation mechanisms. Under the optimum experimental conditions, good linearity was observed with R2 > 0.999. The extraction recoveries were found in the range of 93.9−101.2% with repeatabilities and reproducibilities in 0.89−8.12% and 2.12−10.56%, respectively. The limits of detection ranged from 0.0025 to 0.057 µg/mL. The developed method was applied to commercial polybutylene adipate co-terephthalate (PBAT) biodegradable mulch film samples for analysis of the diverse residual diisocyanates and related diamine additives. The components varied greatly among the sample from different origin. Overall, this study provides a reliable method for assessing safety in biodegradable mulch films.

Conformational flexibility in the enterovirus RNA replication platform.

A presumed RNA cloverleaf (5'CL), located at the 5'-most end of the noncoding region of the enterovirus genome, is the primary established site for initiation of genomic replication. Stem-loop B (SLB) and stem-loop D (SLD), the two largest stem-loops within the 5'CL, serve as recognition sites for protein interactions that are essential for replication. Here we present the solution structure of rhinovirus serotype 14 5'CL using a combination of nuclear magnetic resonance spectroscopy and small-angle X-ray scattering. In the absence of magnesium, the structure adopts an open, somewhat extended conformation. In the presence of magnesium, the structure compacts, bringing SLB and SLD into close contact, a geometry that creates an extensive accessible major groove surface, and permits interaction between the proteins that target each stem-loop.

Author Correction: Mitochondrial metabolism promotes adaptation to proteotoxic stress.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mitochondrial metabolism promotes adaptation to proteotoxic stress.

The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but are key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome-inhibitor resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small molecule compound elesclomol. Genome-wide CRISPR-Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and nuclear-magnetic-resonance-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies explain a fundamental mechanism by which cells adapt to proteotoxic stress and suggest strategies to mitigate proteasome inhibitor resistance.

Comparative transcriptome and metabolomic profiling reveal the complex mechanisms underlying the developmental dynamics of tobacco leaves.

Although the leaf is the most important photosynthetic organ in most plants, many of the molecular mechanisms underlying leaf developmental dynamics remain to be explored. To better understand the transcriptional regulatory mechanisms involved in leaf development, we conducted comparative transcriptomic and metabolomic analysis of leaves from seven positions on tobacco (Nicotiana tabacum) plants. A total of 35,622 unique differentially expressed genes and 79 metabolites were identified. A time-series expression analysis detected two interesting transcriptional profiles, one comprising 10,197 genes that displayed continual up-regulation during leaf development and another comprising 4696 genes that displayed continual down-regulation. Combining these data with co-expression network results identified four important regulatory networks involved in photorespiration and the tricarboxylic acid cycle; these networks may regulate carbon/nitrogen balance during leaf development. We also found that the transcription factor NtGATA5 acts as a hub associated with C and N metabolism and chloroplast development during leaf development through regulation of phytohormones. Furthermore, we investigated the transcriptional dynamics of genes involved in the auxin, cytokinin, and jasmonic acid biosynthesis and signaling pathways during tobacco leaf development. Overall, our study greatly expands the understanding of the regulatory network controlling developmental dynamics in plant leaves.

Dexamethasone Versus Prednisone or Prednisolone for Acute Pediatric Asthma Exacerbations in the Emergency Department: A Meta-Analysis.

OBJECTIVE: This study evaluates the efficacy and tolerability of dexamethasone (DEX) as an alternative to prednisone/prednisolone (PRED) for the treatment of pediatric asthma exacerbations in emergency department (ED). METHODS: Fixed-effects meta-analyses of selected endpoints were performed by using data taken from relevant studies identified by following a priori eligibility criteria after a comprehensive literature search in several electronic databases. RESULTS: Data from 10 studies (3208 pediatric asthma patients [1616 DEX treated and 1592 PRED treated], 4.77 years [95% confidence interval, 3.80-5.56 years], 63% [57.76%-62.68%] males) were used. Risk of vomiting drug was significantly lower in DEX group than in PRED group (risk ratio, 0.29 [0.18-0.48]; P ˂ 0.00001). Emergency department stay between DEX and PRED treated patients was statistically different (0.16 [0.03-0.40] hours; P = 0.02) but may not be clinically meaningful. The number of ß-agonist therapies received by DEX- and PRED-treated patients was similar. Treatments with both DEX and PRED were associated with improvement in asthma status assessment scores, and there was no significant difference between the groups. There were also no differences between the groups in hospitalization rate, ED revisit rate, and hospital admission rate after relapse. CONCLUSIONS: Dexamethasone is a suitable alternative to PRED for the treatment of pediatric asthma exacerbation in ED.

ISCU interacts with NFU1, and ISCU[4Fe-4S] transfers its Fe-S cluster to NFU1 leading to the production of holo-NFU1.

NFU1 is a late-acting factor in the biogenesis of human mitochondrial iron-sulfur proteins. Mutations in NFU1 are associated with genetic diseases such as multiple mitochondrial dysfunctions syndrome 1 (MMDS1) that involve defects in mitochondrial [4Fe-4S] proteins. We present results from NMR spectroscopy, small angle X-ray scattering, size exclusion chromatography, and isothermal titration calorimetry showing that the structured conformer of human ISCU binds human NFU1. The dissociation constant determined by ITC is Kd = 1.1 ± 0.2 µM. NMR and SAXS studies led to a structural model for the complex in which the cluster binding region of ISCU interacts with two α-helices in the C-terminal domain of NFU1. In vitro experiments demonstrate that ISCU[4Fe-4S] transfers its Fe-S cluster to apo-NFU1, in the absence of a chaperone, leading to the assembly of holo-NFU1. By contrast, the cluster of ISCU[2Fe-2S] remains bound to ISCU in the presence of apo-NFU1.

Miniature Hollow Gold Nanorods with Enhanced Effect for In Vivo Photoacoustic Imaging in the NIR-II Window.

The miniaturization of gold nanorods exhibits a bright prospect for intravital photoacoustic imaging (PAI) and the hollow structure possesses a better plasmonic property. Herein, miniature hollow gold nanorods (M-AuHNRs) (≈46 nm in length) possessing strong plasmonic absorbance in the second near-infrared (NIR-II) window (1000-1350 nm) are developed, which are considered as the most suitable range for the intravital PAI. The as-prepared M-AuHNRs exhibit 3.5 times stronger photoacoustic signal intensity than the large hollow Au nanorods (≈105 nm in length) at 0.2 optical density under 1064 nm laser irradiation. The in vivo biodistribution measurement shows that the accumulation in tumor of miniature nanorods is twofold as high as that of the large counterpart. After modifying with a tumor-targeting molecule and fluorochrome, in living tumor-bearing mice, the M-AuHNRs group gives a high fluorescence intensity in tumors, which is 3.6-fold that of the large ones with the same functionalization. Moreover, in the intravital PAI of living tumor-bearing mice, the M-AuHNRs generate longer-lasting and stronger photoacoustic signal than the large counterpart in the NIR-II window. Overall, this study presents the fabrication of M-AuHNRs as a promising contrast agent for intravital PAI.

Template-induced Al distribution in MOR and enhanced activity in dimethyl ether carbonylation.

As the activity of dimethyl ether (DME) carbonylation over mordenite proportionally correlates with the Brønsted acid sites (BAS) in 8-membered ring (8-MR), enhancing the concentration of BAS in the 8-MR of MOR is important to improve the efficiency of the reaction. Herein, we report that the distribution of the BAS in the zeolite catalyst H-MOR can be altered by the synthesis of H-MOR with different cyclic amine structure-directing templates, several of which have not been reported previously for MOR synthesis. By combining FTIR, ICP, TG analysis and DFT calculations, it is verified that the strength of the interaction between amine or sodium cations and [AlO4]- in the zeolite framework plays a decisive role in Al distribution, owing to the competitive effect between Na+ and the cyclic amine compensating negative charges from the framework [AlO4]-. Quantitative analysis of the BAS in the 12-MR and 8-MR identifies the optimum template for maximizing the BAS in the 8-MR. It is shown that the enhanced activity of the H-MOR for the DME carbonylation to methyl acetate correlates with the increase in the BAS in the 8-MR. Our finding thus provides a facile strategy to direct Al location within different channels of the zeolite, which must benefit spatially confined reaction systems.

Transcriptome analysis on responses of orchardgrass (Dactylis glomerata L.) leaves to a short term flooding.

BACKGROUND: Orchardgrass (Dactylis glomerata L.) is a popular cool-season perennial grass with a high production value, and orchardgrass seed is the fourth top-selling forage grass seed in the world. However, its yield and quality are often affected by flooding. To date, the molecular responses of orchardgrass to flooding were poorly understood. RESULTS: Here, we performed mRNA-seq to explore the transcriptomic responses of orchardgrass to a short term flooding (8 h and 24 h). There were 1454 and 565 differentially expressed genes identified in the 8 h and 24 h of flooding, respectively, compared to well control. GO functional enrichment analysis showed that oxidoreductase activity and oxidation-reduction process were highly present, suggesting that flooding induced the response to oxygen stress. Pathways enrichment analysis highlights the importance of glutathione metabolism, peroxidase, glycolysis and plant hormone signal transduction in response to flooding acclimation. Besides, the ROS clearance system is activated by significantly expressed glutathione S-transferase and genes encoding SOD and CAT (CAT1 and CDS2). The significant positive correlation between RNA sequencing data and a qPCR analysis indicated that the identified genes were credible. CONCLUSION: In the process of orchardgrass response to flooding stress, multiple differential genes and biological processes have participated in its acclimation to flooding, especially the biological processes involved in the removal of ROS. These results provide a basis for further research on the adaptation mechanism of orchardgrass to flood tolerance.

A New Type of Capping Agent in Nanoscience: Metal Cations.

Capping agents are the essential factor in nanoscience and nanotechnology. However, the types of capping agents are greatly limited. Defying conventional beliefs, here is shown that metal cations can also be considered as capping agents for oxide nanoparticles, particularly in maintaining their colloidal stability and controlling their facets. Here the general stabilizing effects of multivalent cations for oxide nanoparticles, and the facet controlling role of Al3+ ions in the growth and ripening of Cu2 O octahedra, are demonstrated. This discovery broadens the view of capping agent and opens doors for nanosynthesis, surface treatment, and beyond.