EMC rectifies the topology of multipass membrane proteins.

Most eukaryotic multipass membrane proteins are inserted into the membrane of the endoplasmic reticulum. Their transmembrane domains (TMDs) are thought to be inserted co-translationally as they emerge from a membrane-bound ribosome. Here we find that TMDs near the carboxyl terminus of mammalian multipass proteins are inserted post-translationally by the endoplasmic reticulum membrane protein complex (EMC). Site-specific crosslinking shows that the EMC's cytosol-facing hydrophilic vestibule is adjacent to a pre-translocated C-terminal tail. EMC-mediated insertion is mostly agnostic to TMD hydrophobicity, favored for short uncharged C-tails and stimulated by a preceding unassembled TMD bundle. Thus, multipass membrane proteins can be released by the ribosome-translocon complex in an incompletely inserted state, requiring a separate EMC-mediated post-translational insertion step to rectify their topology, complete biogenesis and evade quality control. This sequential co-translational and post-translational mechanism may apply to ~250 diverse multipass proteins, including subunits of the pentameric ion channel family that are crucial for neurotransmission.

Mechanism of signal-anchor triage during early steps of membrane protein insertion.

Most membrane proteins use their first transmembrane domain, known as a signal anchor (SA), for co-translational targeting to the endoplasmic reticulum (ER) via the signal recognition particle (SRP). The SA then inserts into the membrane using either the Sec61 translocation channel or the ER membrane protein complex (EMC) insertase. How EMC and Sec61 collaborate to ensure SA insertion in the correct topology is not understood. Using site-specific crosslinking, we detect a pre-insertion SA intermediate adjacent to EMC. This intermediate forms after SA release from SRP but before ribosome transfer to Sec61. The polypeptide's N-terminal tail samples a cytosolic vestibule bordered by EMC3, from where it can translocate across the membrane concomitant with SA insertion. The ribosome then docks on Sec61, which has an opportunity to insert those SAs skipped by EMC. These results suggest that EMC acts between SRP and Sec61 to triage SAs for insertion during membrane protein biogenesis.

DNAzyme recognition triggered cascade signal amplification for rapid and highly sensitive visual detection of uranyl ions.

This work presents a rapid and highly sensitive colorimetric assay using bifunctional DNA probe decorated agarose microbeads (MBs) coupled with a cascade signal amplification system, including rolling circle amplification (RCA) and the hemin/G-quadruplex-catalyzed colorimetric reaction, for visualized detection of uranyl ions. The DNA probe integrates the UO22+-specific DNAzyme/substrate as the target recognition unit and a DNA primer as the signal conversion unit. The presence of uranyl ions induces the efficient cleavage of the DNA substrates with the catalysis of DNAzyme. Then the conjugated primers are released from MBs, initiating the RCA reaction (the first amplification). The RCA product consists of repetitive G-quadruplexes that can lead to a second amplification by catalyzing the oxidation of ABTS2- with hemin binding, resulting in a coloration that is visible to the naked eye. The whole assay procedure could be finished within 40 min, including recognition of uranyl and DNA cleavage (5 min), the RCA reaction (30 min) and data readout either by eye or using a UV-vis spectrometer (5 min for each sample). In the optimal conditions, concentrations as low as 5 nM uranyl ions could be distinguished by the naked eye. With UV-vis spectrometric measurement, the visible absorbance had a linear relationship with the concentration of uranyl ions with a dynamic range from 1 nM to 50 nM, and a low detection limit of 0.48 nM (i.e. ∼0.12 ppb) was obtained. Excellent selectivity and anti-interference capability in water samples were also certified. This facile visualized assay could be applied in detecting trace-level uranium for on-site environmental analysis.

Cloud point extraction associated with differential pulse voltammetry: preconcentration and determination of trace uranyl in natural water.

A procedure for the electroanalytical determination of uranyl ions pre-concentrated from natural water by cloud point extraction (CPE) is developed in this study. CPE parameters, such as surfactant concentration, extractant concentration, pH and additive concentration were optimized. After CPE, the solution was diluted for electrochemical determination by differential pulse voltammetry (DPV) with a mercury film electrode (Hg-GCE). The current response of uranyl showed a linear relationship with concentration from 10 nmol L-1 to 1 µmol L-1. The hyphenated method combining CPE and DPV achieved a detection limit of uranyl as low as 0.15 nmol L-1. The presence of some foreign ions interfered greatly with the current response of electrochemical detection. Therefore, the hyphenated technique combining CPE and DPV is important because the CPE step provides selectivity against the co-existing metal ions for electrochemical detection. No interference was seen from the representative foreign metal ions in the CPE-DPV method. The developed method was successfully applied for the determination of uranyl ions in natural water. The average recovery using CPE-DPV in real samples varied from 94.4% to 103.2% and the precision was comparable with that of inductively coupled plasma mass spectrometry (ICP-MS), indicating the good accuracy and precision of the method developed. This hyphenated technique could have greater potential applications for the determination of uranyl ions in aqueous environments.

Reticulon-3 Promotes Endosome Maturation at ER Membrane Contact Sites.

ER tubules form and maintain membrane contact sites (MCSs) with endosomes. How and why these ER-endosome MCSs persist as endosomes traffic and mature is poorly understood. Here we find that a member of the reticulon protein family, Reticulon-3L (Rtn3L), enriches at ER-endosome MCSs as endosomes mature. We show that this localization is due to the long divergent N-terminal cytoplasmic domain of Rtn3L. We found that Rtn3L is recruited to ER-endosome MCSs by endosomal protein Rab9a, which marks a transition stage between early and late endosomes. Rab9a utilizes an FSV region to recruit Rtn3L via its six LC3-interacting region motifs. Consistent with our localization results, depletion or deletion of RTN3 from cells results in endosome maturation and cargo sorting defects, similar to RAB9A depletion. Together our data identify a tubular ER protein that promotes endosome maturation at ER MCSs.

Stress sensor Ire1 deploys a divergent transcriptional program in response to lipid bilayer stress.

Membrane integrity at the endoplasmic reticulum (ER) is tightly regulated, and its disturbance is implicated in metabolic diseases. Using an engineered sensor that activates the unfolded protein response (UPR) exclusively when normal ER membrane lipid composition is compromised, we identified pathways beyond lipid metabolism that are necessary to maintain ER integrity in yeast and in C. elegans. To systematically validate yeast mutants that disrupt ER membrane homeostasis, we identified a lipid bilayer stress (LBS) sensor in the UPR transducer protein Ire1, located at the interface of the amphipathic and transmembrane helices. Furthermore, transcriptome and chromatin immunoprecipitation analyses pinpoint the UPR as a broad-spectrum compensatory response wherein LBS and proteotoxic stress deploy divergent transcriptional UPR programs. Together, these findings reveal the UPR program as the sum of two independent stress responses, an insight that could be exploited for future therapeutic intervention.

Endoplasmic reticulum contact sites regulate the dynamics of membraneless organelles.

Tethered interactions between the endoplasmic reticulum (ER) and other membrane-bound organelles allow for efficient transfer of ions and/or macromolecules and provide a platform for organelle fission. Here, we describe an unconventional interface between membraneless ribonucleoprotein granules, such as processing bodies (P-bodies, or PBs) and stress granules, and the ER membrane. We found that PBs are tethered at molecular distances to the ER in human cells in a tunable fashion. ER-PB contact and PB biogenesis were modulated by altering PB composition, ER shape, or ER translational capacity. Furthermore, ER contact sites defined the position where PB and stress granule fission occurs. We thus suggest that the ER plays a fundamental role in regulating the assembly and disassembly of membraneless organelles.

Flexible and adhesive tape decorated with silver nanorods for in-situ analysis of pesticides residues and colorants.

A flexible adhesive tape decorated with SERS-active silver nanorods (AgNRs) in the form of an array nanostructure is described. The tape was constructed by transferring the AgNRs nanostructures from silicon to the transparent tape by a "paste & peel off" procedure. The transparent, sticky, and flexible properties of commercial tapes allow almost any SERS-inactive irregular surface to be detected in-situ by pasting the SERS tape onto the position to be analyzed. Three examples for an analytical application are presented, viz. determination of (a) tetramethylthiuram disulfide and thiabendazole (two pesticides), (b) colorants in the gel of a writing pen, and (c) the fluorophore Rhodamine B. The tetramethylthiuram disulfide on apple surface was rapidly detected with a LOD of 28.8 ng·cm-2. The AgNRs effectively quenched the fluorescence of the matrix and fluorophores, this enabling the colorants and Rhodamine B to be identified. The results demonstrated that the SERS tape can be used for versatile in-situ detection. Conceivably, it may find applications in food analysis, non-invasive identification, environmental monitoring, and in other areas of daily life. Graphic abstract A flexible and adhesive SERS active tape decorated with silver nanorods (AgNRs) arrays was constructed through a "paste & peel off" method. It can be used as a versatile in situ analysis platform for various applications.

Graphene Oxide Carburization Enhanced Ionization Efficiency for TIMS Isotope Ratio Analysis of Uranium at Trace Level.

Isotope analysis of trace uranium is important in nuclear safeguards and nuclear forensics, which requires the analytical methodologies with high sensitivity, accuracy, and precision. As one of the most powerful techniques in isotopic measurement, thermal ionization mass spectrometry (TIMS) usually suffers from its relatively low sensitivity in ultratrace measurements. To overcome this limitation, we have developed a new filament carburization technique for TIMS, with graphene oxide (GO) as the ionization enhancer. A high and steady ionization efficiency of ∼0.2% for uranium was achieved in single-filament mode, which was 10× the classical double-filament method. With total evaporation (TE) measurements, this method was validated with certified reference materials (CRMs) at the picogram level, and the relative uncertainties for n(235U)/ n(238U) were as low as the ∼1% level. The enhancement mechanism of GO's promoting effect on uranium ionization was attributed to the uniform microstructure facilitating energy transfer and formation of carbides. This approach provides an alternative simple and rapid method for trace uranium isotope analysis with high sensitivity and excellent repeatability. Filament carburization and uranium loading could be accomplished within 10 min. This technique has great advantage in analysis of trace uranium isotope ratios and can be applied in the researches of environmental analysis and nuclear forensics.

Reduced graphene oxide nanosheets modified with plasmonic gold-based hybrid nanostructures and with magnetite (Fe3O4) nanoparticles for cyclic voltammetric determination of arsenic(III).

The authors have fabricated reduced graphene oxide nanosheets (rGO) supported with Fe3O4 nanoparticles and Ag/Au hollow nanoshells. The material was placed on a glassy carbon electrode which is shown to enable highly sensitive determination of As(III) which is first preconcentrated from solution at a potential of -0.35 V (versus Ag/AgCl) for 100 s. The electrode, typically operated at a working potential as low as 0.06 V, has a linear response in the 0.1 to 20 ppb As(III) concentration range and a 0.01 ppb detection limit. The electrochemical sensitivity is 52 µA ppb-1. The high sensitivity is assumed to be the result of various synergistic effects. The method was applied to ultratrace (0.1 ppt) determination of As(III) in real water samples. Graphical abstract The hybrid displays a wide linear response in the 0.1 to 20 ppb As(III) concentration range and a 0.01 ppb detection limit. The high sensitivity is attributed to various synergistic effects. The method was applied to ultratrace determination of As(III) in real water samples.

The contribution of photoinduced charge-transfer enhancement to the SERS of uranyl(VI) in a uranyl-Ag2O complex.

Charge-transfer (CT) is an important enhancement mechanism in the field of surface-enhanced Raman scattering (SERS) that typically increases the Raman intensity of molecules by as much as 10-100 times. Herein, a low-cost Ag2O aggregates substrate was prepared via a facile chemical precipitation method, and the calculated CT-based enhancement factor of the uranyl ions adsorbed on it reached as high as 105, a metal-comparable value. The efficient photoinduced CT process from the valence band of Ag2O to the LUMO of uranyl ions under appropriate excitation sources resulted in the repulsion of the axial oxygen atoms of the OUO bond, which enhanced its polarizability, creating a more intense Raman mode. To the best of our knowledge, this study firstly reports such a strong photoinduced CT enhancement of uranyl ions, with concentrations of 10-8 mol L-1 or lower being detected using this Ag2O substrate. Most importantly, this research has shown that the photoinduced CT enhancement also contributes to the SERS of uranyl ions on pure Ag substrates which have often been ascribed to the electromagnetic enhancement in previous studies. In addition, Ag2O can be used to selectively detect uranyl ions without interference from many other molecules or ions because of the energy matching rule of the photoinduced CT process, which was readily available for uranyl detection in the environmental aqueous solution.

Rapid and sensitive detection of uranyl ion with citrate-stabilized silver nanoparticles by the surface-enhanced Raman scattering technique.

Uranium contamination poses a huge threat to human health due to its widespread use in the nuclear industry and weapons. We proposed a simple and convenient wet-state SERS method for uranyl detection based on the citrate-stabilized silver nanoparticles. The effect of citrate on the detection performance was also discussed. By using the citrate as an internal reference to normalize the peak of uranyl, a quantitative analysis was achieved and a good linear relationship of uranyl concentration from 0.2 to 5 µM with the limit of detection of 60 nM was obtained. With its simplicity, convenience and cost-effectiveness, this method has great potential for the detection of other molecules also.

Here, there, and everywhere: The importance of ER membrane contact sites.

Our textbook image of organelles has changed. Instead of revealing isolated cellular compartments, the picture now emerging shows organelles as largely interdependent structures that can communicate through membrane contact sites (MCSs). MCSs are sites where opposing organelles are tethered but do not fuse. MCSs provide a hybrid location where the tool kits of two different organelles can work together to perform vital cellular functions, such as lipid and ion transfer, signaling, and organelle division. Here, we focus on MCSs involving the endoplasmic reticulum (ER), an organelle forming an extensive network of cisternae and tubules. We highlight how the dynamic ER network regulates a plethora of cellular processes through MCSs with various organelles and with the plasma membrane.

Self-assembly of silver nanoparticles as high active surface-enhanced Raman scattering substrate for rapid and trace analysis of uranyl(VI) ions.

A facile surface-enhanced Raman scattering (SERS) substrate based on the self-assembly of silver nanoparticles on the modified silicon wafer was obtained, and for the first time, an advanced SERS analysis method basing on this as-prepared substrate was established for high sensitive and rapid detection of uranyl ions. Due to the weakened bond strength of OUO resulting from two kinds of adsorption of uranyl species ("strong" and "weak" adsorption) on the substrate, the ν1 symmetric stretch vibration frequency of OUO shifted from 871cm-1 (normal Raman) to 720cm-1 and 826cm-1 (SERS) along with significant Raman enhancement. Effects of the hydrolysis of uranyl ions on SERS were also investigated, and the SERS band at ~826cm-1 was first used to approximately define the constitution of uranyl species at trace quantity level. Besides, the SERS intensity was proportional to the variable concentrations of uranyl nitrate ranging from 10-7 to 10-3molL-1 with an excellent linear relation (R2=0.998), and the detection limit was ~10-7molL-1. Furthermore, the related SERS approach involves low-cost substrate fabrication, rapid and trace analysis simultaneously, and shows great potential applications for the field assays of uranyl ions in the nuclear fuel cycle and environmental monitoring.

Multiple dynamin family members collaborate to drive mitochondrial division.

Mitochondria cannot be generated de novo; they must grow, replicate their genome, and divide in order to be inherited by each daughter cell during mitosis. Mitochondrial division is a structural challenge that requires the substantial remodelling of membrane morphology. Although division factors differ across organisms, the need for multiple constriction steps and a dynamin-related protein (Drp1, Dnm1 in yeast) has been conserved. In mammalian cells, mitochondrial division has been shown to proceed with at least two sequential constriction steps: the endoplasmic reticulum and actin must first collaborate to generate constrictions suitable for Drp1 assembly on the mitochondrial outer membrane; Drp1 then further constricts membranes until mitochondrial fission occurs. In vitro experiments, however, indicate that Drp1 does not have the dynamic range to complete membrane fission. In contrast to Drp1, the neuron-specific classical dynamin dynamin-1 (Dyn1) has been shown to assemble on narrower lipid profiles and facilitate spontaneous membrane fission upon GTP hydrolysis. Here we report that the ubiquitously expressed classical dynamin-2 (Dyn2) is a fundamental component of the mitochondrial division machinery. A combination of live-cell and electron microscopy in three different mammalian cell lines reveals that Dyn2 works in concert with Drp1 to orchestrate sequential constriction events that build up to division. Our work underscores the biophysical limitations of Drp1 and positions Dyn2, which has intrinsic membrane fission properties, at the final step of mitochondrial division.

Free-Standing Monolayered Metallic Nanoparticle Networks as Building Blocks for Plasmonic Nanoelectronic Junctions.

The effective coupling of optical surface plasmons (SPs) and electron transport in a plasmonic-electronic device is one of the fundamental issues in nanoelectronics and the emerging field of plasmonics, and offer promise in providing a solution to next generation nanocircuits in which all coupling is in the near field. Attempts toward this end, however, are limited because of the integration challenge to compatible nanodevices. To date, direct electrical detection of SP-electron coupling from metallic nanostructures alone are not reported, and thus it remains a great experimental challenge. In this paper, we succeed in preparing a new suspended-film-type nanoelectronic junction, in which free-standing 2D fractal nanoparticle networks act as plasmonically active nanocomponents. Direct electrical detection of optical collective SPs was evidenced by photocurrent response of the junction upon illumination. Room-temperature I-V characteristics, differing from nonlinear to Ohmic behaviors, are found to be sensitive to the nanometer-scale morphology changes of the nanomembranes. The finding and approach may enable the development of advanced plasmonic nanocircuits and new nanoelectronics, nanophotonics, and (solar) energy applications.

[Design and Fabrication of Silver Nanoparticles/Graphen Complex Substrate and Its Application for Detecting Uranium (â ¥)].

Uranium is one of the important nuclear materials to nuclear industry. Because of the direct disposal of spent fuel, there is still a huge possibility that uranium migrates into the groundwater, causing water contamination. It is of great importance to understand the concentration and their species distribution in aqueous solutions. Surface-Enhanced Raman Scattering (SERS) technique has been widely used for the detection of uranium (Ⅵ). However, the interactions between uranium (Ⅵ) and SERS substrate cause the symmetric stretching vibration peak of uranium (Ⅵ) shift to low wave number direction, which is unfavorable for confirming the species of uranium (Ⅵ) in aqueous solution. For instance, the normal Raman bands of uranyl in nitric acid solution are 871 cm-1, which belongs to the symmetric stretching mode of UO2+2. However, it moves to 710 cm-1 on the surface of silver nanorods SERS substrtate. What's more, different SERS substrate causes different number of shift. Graphene has advantages of inertness and integrity as well as 2-dimensional thickness. In this paper, graphene-isolated SERS substrate which is silver nanoparticles (AgNPs)/graphene complex substrate, was designed to prevent the interaction between SERS substrate and it was analyzed by using the inert graphene layer. First of all, according to our previous work, AgNPs SERS substrate was fabricated on silicon wafer by using an ascorbic acid-actived self-assembly method. Then, AgNPs/graphene complex substrate was prepared by transfering monolayer graphene onto the self-assembly AgNPs substrate. The morphology of complex substrate was obtained by SEM. Some AgNPs link together closely to form nanochain structures. Nanochain structures were distributed evenly on the surface of silicon wafer. The 2-dimensional thickness of graphene did not affect the morphology of AgNPs. When using the complex substrate to detect uranyl nitrate (5×10-4 mol·L-1)，the Raman peak that appeared around 771 cm-1 is considered to be the symmetric stretching mode of UO2+2, shifting back about 52 cm-1 to high wave number direction when compared with AgNPs substrate, which was about ~719 cm-1. The result indicates that graphene layer isolates the interaction between AgNPs substrate and uranyl in some degree.

Smart Plasmonic Glucose Nanosensors as Generic Theranostic Agents for Targeting-Free Cancer Cell Screening and Killing.

Fast and accurate identification of cancer cells from healthy normal cells in a simple, generic way is very crucial for early cancer detection and treatment. Although functional nanoparticles, like fluorescent quantum dots and plasmonic Au nanoparticles (NPs), have been successfully applied for cancer cell imaging and photothermal therapy, they suffer from the main drawback of needing time-consuming targeting preparation for specific cancer cell detection and selective ablation. The lack of a generic and effective method therefore limits their potential high-throughput cancer cell preliminary screening and theranostic applications. We report herein a generic in vitro method for fast, targeting-free (avoiding time-consuming preparations of targeting moiety for specific cancer cells) visual screening and selective killing of cancer cells from normal cells, by using glucose-responsive/-sensitive glucose oxidase-modified Ag/Au nanoshells (Ag/Au-GOx NSs) as a smart plasmonic theranostic agent. The method is generic to some extent since it is based on the distinct localized surface plasmon resonance (LSPR) responses (and colors) of the smart nanoprobe with cancer cells (typically have a higher glucose uptake level) and normal cells.

Ionic liquid-functionalized fluorescent carbon nanodots and their applications in electrocatalysis, biosensing, and cell imaging.

In this article, ionic liquid-functionalized carbon nanodots (IL-CDs) were produced in a simple manner by electrochemical exfoliation of graphite rods in the presence of an amino-terminated ionic liquid, and their preliminary applications were exploited. TEM and AFM results showed that these IL-CDs are about 2.6 nm in diameter. The small-sized IL-CDs have strong photoluminescence, with a quantum yield of about 11.3%, and could be used for cell imaging. Moreover, the IL-CDs exhibit good electron transfer properties and catalytic activities for O2 and H2O2 reduction. Additionally, the as-prepared IL-CDs can be applied as a matrix for immobilizing enzymes (glucose oxidase) to construct biosensors. Due to these favorable properties, IL-CDs will find promising practical applications in electrocatalysis, biosensing, and bioimaging.

Endoplasmic reticulum stress response in yeast and humans.

Stress pathways monitor intracellular systems and deploy a range of regulatory mechanisms in response to stress. One of the best-characterized pathways, the UPR (unfolded protein response), is an intracellular signal transduction pathway that monitors ER (endoplasmic reticulum) homoeostasis. Its activation is required to alleviate the effects of ER stress and is highly conserved from yeast to human. Although metazoans have three UPR outputs, yeast cells rely exclusively on the Ire1 (inositol-requiring enzyme-1) pathway, which is conserved in all Eukaryotes. In general, the UPR program activates hundreds of genes to alleviate ER stress but it can lead to apoptosis if the system fails to restore homoeostasis. In this review, we summarize the major advances in understanding the response to ER stress in Sc (Saccharomyces cerevisiae), Sp (Schizosaccharomyces pombe) and humans. The contribution of solved protein structures to a better understanding of the UPR pathway is discussed. Finally, we cover the interplay of ER stress in the development of diseases.