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The monosubstitution of a deuterium atom at the alpha carbon of the neopentyl ligand was established by the presence of a broad singlet for one proton at sigma 3.19 in its 1HNMR spectrum. The evidence for the synthesis of a highly reactive alkyldiene species which mediates the transformation even in the presence of excess trimethylphosphine was described. The transition-metal alkyldiene complex cab influence the addition of C-H bonds of alkanes across the metal-carbon double bond.

Lipopolysaccharide (LPS), a surface polymer of Gram-negative bacteria, helps bacteria survive in different environments and acts as a virulence determinant of host infection. The O-antigen (Oag) component of LPS exhibits a modal chain-length distribution that is controlled by polysaccharide co-polymerases (PCPs). The molecular basis of the regulation of Oag chain-lengths remains unclear, despite extensive mutagenesis and structural studies of PCPs from Escherichia coli and Shigella. Here, we identified a single mutation (A107P) of the Shigella flexneri WzzBSF, by a random mutagenesis approach, that causes a shortened Oag chain-length distribution in bacteria. We determined the crystal structures of the periplasmic domains of wild-type WzzBSF and the A107P mutant. Both structures form a highly similar open trimeric assembly in the crystals, and show a similar tendency to self-associate in solution. Binding studies by bio-layer interferometry reveal cooperative binding of very short (VS)-core-plus-O-antigen polysaccharide (COPS) to the periplasmic domains of both proteins, but with decreased affinity for the A107P mutant. Our studies reveal that subtle and localized structural differences in PCPs can have dramatic effects on LPS chain-length distribution in bacteria, for example by altering the affinity for the substrate, which supports the role of the structure of the growing Oag polymer in this process. [ABSTRACT FROM AUTHOR]

In this article, we present a microfluidic platform for passive fluid pumping for pump-free perfusion cell culture, cell-based assay, and chemical applications. By adapting the passive membrane-controlled pumping principle from the previously developed perfusion microplate, which utilizes a combination of hydrostatic pressure generated by different liquid levels in the wells and fluid wicking through narrow strips of a porous membrane connecting the wells to generate fluid flow, a series of pump-free membrane-controlled perfusion microfluidic devices was developed and their use for pump-free perfusion cell culture and cell-based assays was demonstrated. Each pump-free membrane-controlled perfusion microfluidic device comprises at least three basic components: an open well for generating fluid flow, a micron-sized deep chamber/channel for cell culture or for fluid connection, and a wettable porous membrane for controlling the fluid flow. Each component is fluidically connected either by the porous membrane or by the micron-sized deep chamber/channel. By adapting and incorporating the passive membrane-controlled pumping principle into microfluidic devices, all the benefits of microfluidic technologies, such as small sample volumes, fast and efficient fluid exchanges, and fluid properties at the micro-scale, can be fully taken advantage of with this pump-free membrane-controlled perfusion microfluidic platform. [ABSTRACT FROM AUTHOR]

The Shigella flexneri autotransporter protein IcsA is essential for intra- and intercellular spread, and icsA mutants are attenuated in several models. However, the pathogenic significance of the outer membrane protease IcsP, which orchestrates the polar distribution of IcsA on the bacterial surface, remains unclear. To further examine this point, we constructed icsP mutants in the two most commonly studied S. flexneri strains and evaluated their in vitro and in vivo performance relative to wild type. Both icsP mutants showed aberrant surface distribution of IcsA, but the in vitro consequences depended upon the cell line being used to assess bacterial motility and plaque formation. Evaluating the behaviour of the mutants in two mouse models suggested functional expression of icsP might limit bacterial persistence and the associated inflammation in host tissues, consistent with the findings in one of the three cell lines used. [ABSTRACT FROM AUTHOR]

Shigella flexneri implements the Wzy-dependent pathway to biosynthesize the O antigen (Oag) component of its surface lipopolysaccharide. The inner membrane polymerase WzySF catalyzes the repeat addition of undecaprenol-diphosphate-linked Oag (Und-PP-RUs) to produce a polysaccharide, the length of which is tightly regulated by two competing copolymerase proteins, WzzSF (short-type Oag; 10 to 17 RUs) and WzzpHS-2 (very-long-type Oag; >90 RUs). The nature of the interaction between WzySF and WzzSF/WzzpHS-2 in Oag polymerization remains poorly characterized, with the majority of the literature characterizing the individual protein constituents of the Wzy-dependent pathway. Here, we report instead a major investigation into the specific binding interactions of WzySF with its copolymerase counterparts. For the first time, a region of WzySF that forms a unique binding site for WzzpHS-2 has been identified. Specifically, this work has elucidated key WzySF moieties at the N- and C-terminal domains (NTD and CTD) that form an intramolecular pocket modulating the WzzpHS-2 interaction. Novel copurification data highlight that disruption of residues within this NTD-CTD pocket impairs the interaction with WzzpHS-2 without affecting WzzSF binding, thereby specifically disrupting polymerization of longer polysaccharide chains. This study provides a novel understanding of the molecular interaction of WzySF with WzzSF/WzzpHS-2 in the Wzy-dependent pathway and, furthermore, detects the Wzy/Wzz/Und-PP-Oag complex for the first time. Beyond S. flexneri, this work may be extended to provide insight into the interactions between protein homologues expressed by related species, especially members of Enterobacteriaceae, that produce dual Oag chain length determinants. [ABSTRACT FROM AUTHOR]

Research into the thermal chemistry of Cp*W(NO)(CH2CMe3)2 in various solvents is presented. It was established that additional C-H activation chemistry can arise depending on the nature of the solvent used.

Transport of macromolecules between the cytoplasm and the nucleus is critical for the function of all eukaryotic cells. Large macromolecular channels termed nuclear pore complexes that span the nuclear envelope mediate the bidirectional transport of cargoes between the nucleus and cytoplasm. However, the influence of macromolecular trafficking extends past the nuclear pore complex to transcription and RNA processing within the nucleus and signaling pathways that reach into the cytoplasm and beyond. At the Mechanisms of Nuclear Transport biennial meeting held from October 18 to 23, 2013 in Woods Hole, MA, researchers in the field met to report on their recent findings. The work presented highlighted significant advances in understanding nucleocytoplasmic trafficking including how transport receptors and cargoes pass through the nuclear pore complex, the many signaling pathways that impinge on transport pathways, interplay between the nuclear envelope, nuclear pore complexes, and transport pathways, and numerous links between transport pathways and human disease. The goal of this review is to highlight newly emerging themes in nuclear transport and underscore the major questions that are likely to be the focus of future research in the field. [ABSTRACT FROM AUTHOR]

L7536 --> 787.13 --> Dbp2 is a DEAD‐box RNA helicase in S. cerevisiae, whose main function is connected to the ribosomal RNA processing and transcription. Interestingly, the localization of Dbp2 is also highly dependent on the presence of glucose, which enables us to study how cellular stress impacts gene expression machinery. Studies from the Tran laboratory show that the rapid nuclear export of Dbp2 in the absence of glucose. Furthermore, unpublished studies from the Tran lab have linked specific phosphorylation events on Dbp2 to glucose‐dependent localization. To determine if phosphorylation affects Dbp2's function and localization, our first step is to generate mutant Dbp2 proteins that localize to the nucleus or the cytoplasm, irrespective of the presence of glucose. To this end, site‐directed mutagenesis was used on plasmids containing wild type DBP2 genes to convert codons encoding select serine residues to codons that would encode amino acids mimicking the dephosphorylated and phosphorylated state of Dbp2. Following transformation of these mutant‐expressing plasmids and a wild type DBP2 as a control into a dbp2∆strain, a serial dilution spot assay was performed on glucose and galactose plates (mimicking – glucose). We observed that both the phosphorylation mutants (both the non‐phosphorylatable and the phosphor‐mimetic) grew substantially better than our wild type Dbp2 and empty vector. This was unexpected and will be followed up with future studies in the Tran laboratory. In addition to mutant Dbp2 generation, we also set out to identify protein interaction partners of Dbp2 in the cytoplasm. Our goal was to use proximity‐dependent biotin identification (BioID) using an AirID biotin ligase tag to identify Dbp2's cytoplasmic interaction partners. Following unsuccessful isolation of proteins, immunofluorescence was performed to verify the localization of the ligase tag. Immunofluorescence indicates the AirID tag is more cytoplasmically localized in comparison to nuclear Dbp2, thus, the study was inconclusive. Due to the mislocalization of the AirID tag, in future studies we plan to use different versions of the biotin ligase tag such as the TurboID tag. [ABSTRACT FROM AUTHOR]