Co-translational cross-linking partners of SfmC.

<p>(<b>A</b>) RNCs of SfmC, 126 amino acids in length (<i>arrow</i>), carrying pBpa in their signal sequence either at position I12 or Y14 were synthesized by the PURE system (<i>SfmC-126-I12pBpa</i> and SfmC-126-Y14pBpa). <i>Ffh, FtsY, SecA</i>, and SecYEG proteoliposomes (<i>PL</i>) were present during synthesis as indicated. <i>Asterisks</i>, cross-links to Ffh (x Ffh); <i>arrow heads</i>, cross-links to SecA (x SecA). <i>IPα</i>, immunoprecipitation using the antibodies indicated. (<b>B</b>) as in (A), showing results obtained with SfmC-126 RNCs having pBpa incorporated at V58 located 35 residues downstream of the signal sequence cleavage site (<i>SfmC-126-V58pBpa</i>). <i>Dot</i>, cross-link to SecY (x SecY).</p

The precursors of SfmC and TorT harbouring hydrophobic signal sequences require SRP and FtsY in addition to SecA for maximal translocation.

<p>(<b>A</b>) The ³⁵[S]-labeled precursors of SfmC (<i>pSfmC</i>), TorT (<i>pTorT</i>), and OmpA (<i>pOmpA</i>) were synthesized by the PURE system in the presence of SecYEG-containing proteoliposomes (<i>PL</i>) and purified <i>SecA, Ffh, FtsY</i> proteins (1 μg each) as indicated. All components were mixed on ice prior to starting reactions by incubation at 37°C for 1 h. Radiolabeled translation products were separated by SDS-PAGE and are displayed by phosphorimaging. Translocation into the proteoliposomes is indicated by the relative amount of each precursor transformed into a proteinase K (<i>PK</i>)-resistant species as determined by measuring the intensities of the corresponding bands using ImageQuant 5.2 (GE-Healthcare). Mean values obtained from three independent experiments and standard errors of the means are given. (<b>B</b>) as in (A), except that Ffh, FtsY, and isolated 4.5S RNA were added individually as indicated.</p

Amino acid sequence of signal sequences used.

<p>Amino acid sequence of signal sequences used.</p

Purification of Ffh, FtsY, SecA and SecYEG complex.

<p>His-tagged variants of Ffh, FtsY, and SecA were over-expressed in <i>E. coli</i> and purified from cell extracts by metal affinity chromatography. The SecYEG complex was purified by metal affinity chromatography using a DDM-solubilized membrane pellet obtained from a SecY^HisEG-over-producing <i>E. coli</i> strain. Purified proteins were displayed by SDS-PAGE and staining with Coomassie Blue.</p

Primers used.

<p>Primers used.</p

Cooperative Characterization of <i>In Situ</i> TEM and Cantilever-TGA to Optimize Calcination Conditions of MnO₂ Nanowire Precursors

Calcination plays a vital role during material preparation. However, the calcination conditions have often been determined empirically or have been based on trial and error. Herein we present a cooperative characterization approach to optimize calcination conditions by gas-cell in situ TEM in collaboration with microcantilever-based thermogravimetric analysis (cantilever-TGA) techniques. The morphological evolution of precursors under atmospheric conditions is observed with in situ TEM, and the right calcination temperature is provided by cantilever-TGA. The proposed approach successfully optimizes the calcination conditions of fragile MnO2 nanowire precursors with multiple valence products. The cantilever-TGA shows that a calcination temperature above 560 °C is required to transform the MnO2 precursor to Mn3O4 under an N2 atmosphere, but the in situ TEM indicates that the nanowire structure is destroyed within only 30 min under calcination conditions. Our method further suggests that heating the precursor at 400 °C using an H2-containing atmosphere can produce Mn3O4 nanowires with good electrical properties

Additional file 1 of Association between the neutrophil-to-lymphocyte ratio and in-hospital mortality in patients with chronic kidney disease and coronary artery disease in the intensive care unit

Additional file 1: Materials S1. R code for feature selection

<i>In Situ</i> Molecular Imaging of the Biofilm and Its Matrix

Molecular mapping of live biofilms at submicrometer resolution presents a grand challenge. Here, we present the first chemical mapping results of biofilm extracellular polymeric substance (EPS) in biofilms using correlative imaging between super resolution fluorescence microscopy and liquid time-of-flight secondary ion mass spectrometry (TOF-SIMS). <i>Shewanella oneidensis</i> is used as a model organism. Heavy metal chromate (Cr₂O₇^2–) anions consisting of chromium Cr­(VI) was used as a model environmental stressor to treat the biofilms. Of particular interest, biologically relevant water clusters have been first observed in the biofilms. Characteristic fragments of biofilm matrix components such as proteins, polysaccharides, and lipids can be spatially imaged. Furthermore, characteristic fatty acids (e.g., palmitic acid), quinolone signal, and riboflavin fragments were found to respond after the biofilm is treated with Cr­(VI), leading to biofilm dispersal. Significant changes in water clusters and quorum sensing signals indicative of intercellular communication in the aqueous environment were observed, suggesting that they might result in fatty acid synthesis and inhibition of riboflavin production. The Cr­(VI) reduction seems to follow the Mtr pathway leading to Cr­(III) formation. Our approach potentially opens a new avenue for mechanistic insight of microbial community processes and communications using <i>in situ</i> imaging mass spectrometry and super resolution optical microscopy

In Situ Mass Spectrometric Determination of Molecular Structural Evolution at the Solid Electrolyte Interphase in Lithium-Ion Batteries

Dynamic structural and chemical evolution at solid–liquid electrolyte interface is always a mystery for a rechargeable battery due to the challenge to directly probe a solid–liquid interface under reaction conditions. We describe the creation and usage of in situ liquid secondary ion mass spectroscopy (SIMS) for the first time to directly observe the molecular structural evolution at the solid–liquid electrolyte interface for a lithium (Li)-ion battery under dynamic operating conditions. We have discovered that the deposition of Li metal on copper electrode leads to the condensation of solvent molecules around the electrode. Chemically, this layer of solvent condensate tends to be depleted of the salt anions and with reduced concentration of Li⁺ ions, essentially leading to the formation of a lean electrolyte layer adjacent to the electrode and therefore contributing to the overpotential of the cell. This observation provides unprecedented molecular level dynamic information on the initial formation of the solid electrolyte interphase (SEI) layer. The present work also ultimately opens new avenues for implanting the in situ liquid SIMS concept to probe the chemical reaction process that intimately involves solid–liquid interface, such as electrocatalysis, electrodeposition, biofuel conversion, biofilm, and biomineralization

In Situ Mass Spectrometric Determination of Molecular Structural Evolution at the Solid Electrolyte Interphase in Lithium-Ion Batteries

Dynamic structural and chemical evolution at solid–liquid electrolyte interface is always a mystery for a rechargeable battery due to the challenge to directly probe a solid–liquid interface under reaction conditions. We describe the creation and usage of in situ liquid secondary ion mass spectroscopy (SIMS) for the first time to directly observe the molecular structural evolution at the solid–liquid electrolyte interface for a lithium (Li)-ion battery under dynamic operating conditions. We have discovered that the deposition of Li metal on copper electrode leads to the condensation of solvent molecules around the electrode. Chemically, this layer of solvent condensate tends to be depleted of the salt anions and with reduced concentration of Li⁺ ions, essentially leading to the formation of a lean electrolyte layer adjacent to the electrode and therefore contributing to the overpotential of the cell. This observation provides unprecedented molecular level dynamic information on the initial formation of the solid electrolyte interphase (SEI) layer. The present work also ultimately opens new avenues for implanting the in situ liquid SIMS concept to probe the chemical reaction process that intimately involves solid–liquid interface, such as electrocatalysis, electrodeposition, biofuel conversion, biofilm, and biomineralization