Modulation of Rab GTPase function by a protein phosphocholine transferase.

The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC-domain-containing proteins can alter host-cell functions

Investigation of the twin-arginine translocation pathway in mycobacteria

Mycobacterium tuberculosis is an intracellular pathogen that resides primarily in host macrophages. Proteins that reside on the surface of the bacillus or are secreted into environment are ideally positioned to interact with host cell components and are therefore candidate virulence factors and immunogenic antigens. These proteins are actively transported from the cytoplasm to the cell envelope or extracellular space by specific export pathways. In this work, we describe the initial characterization of the twin-arginine translocation (Tat) export pathway in mycobacteria. The Tat pathway exports folded proteins, including virulence factors, in a number of bacteria. Proteins targeted for Tat-export carry N-terminal signal sequences that have a conserved twin-arginine motif, referred to as R-R-x-φ-φ(φ = uncharged residue). We provide evidence that the Tat pathway is functional in Mycobacterium smegmatis and required for the export of mycobacterial β-lactamases, including BlaC of M. tuberculosis. We demonstrate that BlaC can be used as a reporter to exclusively identify Tat-exported fusion proteins that promote resistance to the β-lactam antibiotic carbenicillin. We describe using the BlaC reporter with a M. tuberculosis genomic expression library to identify random Tat signal sequence-BlaC fusions that promote Tat export in M. smegmatis. Using this approach, we identified eleven M. tuberculosis Tat signal sequences shown to direct the export of the BlaC reporter through the Tat pathway, including the known phospholipase C virulence factors. In addition, we identified a protein lacking the typical twin-arginine motif, suggesting that our current understanding of what defines a Tat signal sequence is limited, and demonstrating the effectiveness of our experimental system. We describe ongoing evaluation to determine the specificity and requirements for export of the authentic full length M. tuberculosis proteins. Finally, we demonstrate the novel finding that BlaC can be used as a reporter for the identification of proteins exported by M. tuberculosis during growth in macrophages. This may lead to the identification of a subset of proteins exported exclusively within the intracellular environment. This work has important implications in determining the role that protein export plays in M. tuberculosis, as well as increasing the understanding of how the Tat pathway functions in bacteria

Effect of fluorine on near-liquidus phase equilibria of an Fe–Mg rich basalt

Author Posting. © The Author(s), 2012. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Chemical Geology 312-313 (2012): 118-126, doi:10.1016/j.chemgeo.2012.04.015.Volatile species (H2O, CO2, F, Cl, etc) have important effects on the formation and crystallization history of basaltic magmas. Here, we have experimentally investigated the effects of F on phase equilibria of Fe-Mg-rich basalt. Our results show that fluorine has large effects on the liquidus temperature and the chemistry of crystallizing minerals. Compared to the F-free system, addition of ~2 wt.% F moves the olivine-pigeonite liquidus point down ~2 kbar and 95 °C (from 12 kbar, 1375 °C to 10 kbar, 1280 °C). With increasing fluorine concentrations, dramatically increases for both pyroxene and olivine, suggesting that fluorine in basaltic magmas complexes primarily with MgO. Complexing with MgO in the melt decreases its MgO activity, and forces the crystallizing minerals to greater Fe/Mg, and so increases . Models of basalt generation, where the magma is fluorine-rich, need to include the effect of not only water but fluorine on liquidus depression and minerals crystallizing/melting. Our results suggest that fluorine may significantly aid in the petrogenesis of silica-poor, alkali-rich magmas in the Earth and Mars.This work was supported by NASA MFR grant # NNX09AL25G to A.H. Treiman and J. Filiberto, a Lunar and Planetary Institute summer internship to J. Wood, and a Packard fellowship for science and engineering to R. Dasgupta

Damaging variants in FOXI3 cause microtia and craniofacial microsomia

Q1Q1Pacientes con Microtia y Microsomía craneofacialPurpose: Craniofacial microsomia (CFM) represents a spectrum of craniofacial malformations, ranging from isolated microtia with or without aural atresia to underdevelopment of the mandible, maxilla, orbit, facial soft tissue, and/or facial nerve. The genetic causes of CFM remain largely unknown. Methods: We performed genome sequencing and linkage analysis in patients and families with microtia and CFM of unknown genetic etiology. The functional consequences of damaging missense variants were evaluated through expression of wild-type and mutant proteins in vitro. Results: We studied a 5-generation kindred with microtia, identifying a missense variant in FOXI3 (p.Arg236Trp) as the cause of disease (logarithm of the odds = 3.33). We subsequently identified 6 individuals from 3 additional kindreds with microtia-CFM spectrum phenotypes harboring damaging variants in FOXI3, a regulator of ectodermal and neural crest development. Missense variants in the nuclear localization sequence were identified in cases with isolated microtia with aural atresia and found to affect subcellular localization of FOXI3. Loss of function variants were found in patients with microtia and mandibular hypoplasia (CFM), suggesting dosage sensitivity of FOXI3. Conclusion: Damaging variants in FOXI3 are the second most frequent genetic cause of CFM, causing 1% of all cases, including 13% of familial cases in our cohort.https://orcid.org/0000-0003-3822-7780https://orcid.org/0000-0002-0729-6866Revista Internacional - IndexadaA1N

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Improving homology-directed repair efficiency in human stem cells.

The generation of induced pluripotent stem cell models of human disease requires efficient modification of one or both alleles depending on dominant or recessive inheritance of the disease. To faithfully recapitulate many disease variants, the introduction of a single base change is required. The introduction of additional silent mutations designed to prevent re-cutting of the modified allele by Cas9 is not an optimal strategy, particularly for non-coding variants. Here, we developed an improved protocol for efficient engineering of single nucleotide variants in human iPS cells. Using a fluorescent BFP-\u3eGFP assay to monitor the incorporation of a single base pair change, we optimized the protocol to achieve HDR in 70% of unselected human iPS cells. The additive effects of cold shock, a small molecule enhancer of HDR and chemically modified ssODN dramatically shift the bias of repair in favor of HDR, resulting in a seven-fold higher ratio of HDR to NHEJ from 0.5 to 3.7

Current Research at the Connley Caves (35LK50): Late Pleistocene/early Holocene Western Stemmed Tradition Occupations in the Fort Rock Basin, Oregon

The Connley Caves in the Fort Rock Basin of Oregon contain stratified deposits dating to the late Pleistocene/early Holocene transition and a stone tool assemblage characteristic of the Western Stemmed Tradition. This research brief details preliminary results including stratigraphic, geochronological, and cultural information yielded from Cave 4. Ongoing research at the site stands to shed light on WST technological activities, intra- and inter-assemblage variability and geochronology, and Paleoarchaic subsistence strategies of the northern Great Basin