Identification of novel clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene

Clostridium perfringens enterotoxin (CPE) is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe) can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid) in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ~65 kb. Complete sequence analysis of the ~65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm) gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ~65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains. © 2011 Miyamoto et al

The ALMA Survey of 70 μm Dark High-mass Clumps in Early Stages (ASHES). VI. The Core-scale CO Depletion

Studying the physical and chemical properties of cold and dense molecular clouds is crucial for the understanding of how stars form. Under the typical conditions of infrared dark clouds, CO is removed from the gas phase and trapped onto the surface of dust grains by the so-called depletion process. This suggests that the CO-depletion factor (f D ) can be a useful chemical indicator for identifying cold and dense regions (i.e., prestellar cores). We have used the 1.3 mm continuum and C18O (2-1) data observed at the resolution of ∼5000 au in the ALMA Survey of 70 μm Dark High-mass Clumps in Early Stages (ASHES) to construct averaged maps of f D in 12 clumps to characterize the earliest stages of the high-mass star formation process. The average f D determined for 277 of the 294 ASHES cores follows an unexpected increase from the prestellar to the protostellar stage. If we exclude the temperature effect due to the slight variations in the NH3 kinetic temperature among different cores, we explain this result as a dependence primarily on the average gas density, which increases in cores where protostellar conditions prevail. This shows that f D determined in high-mass star-forming regions at the core scale is insufficient to distinguish among prestellar and protostellar conditions for the individual cores and should be complemented by information provided by additional tracers. However, we confirm that the clump-averaged f D values correlate with the luminosity-to-mass ratio of each source, which is known to trace the evolution of the star formation process

Report on ICDP Deep Dust workshops: probing continental climate of the late Paleozoic icehouse–greenhouse transition and beyond

Chamberlin and Salisbury's assessment of the Permian a century ago captured the essence of the period: it is an interval of extremes yet one sufficiently recent to have affected a biosphere with near-modern complexity. The events of the Permian - the orogenic episodes, massive biospheric turnovers, both icehouse and greenhouse antitheses, and Mars-analog lithofacies - boggle the imagination and present us with great opportunities to explore Earth system behavior. The ICDP-funded workshops dubbed "Deep Dust," held in Oklahoma (USA) in March 2019 (67 participants from nine countries) and Paris (France) in January 2020 (33 participants from eight countries), focused on clarifying the scientific drivers and key sites for coring continuous sections of Permian continental (loess, lacustrine, and associated) strata that preserve high-resolution records. Combined, the two workshops hosted a total of 91 participants representing 14 countries, with broad expertise. Discussions at Deep Dust 1.0 (USA) focused on the primary research questions of paleoclimate, paleoenvironments, and paleoecology of icehouse collapse and the run-up to the Great Dying and both the modern and Permian deep microbial biosphere. Auxiliary science topics included tectonics, induced seismicity, geothermal energy, and planetary science. Deep Dust 1.0 also addressed site selection as well as scientific approaches, logistical challenges, and broader impacts and included a mid-workshop field trip to view the Permian of Oklahoma. Deep Dust 2.0 focused specifically on honing the European target. The Anadarko Basin (Oklahoma) and Paris Basin (France) represent the most promising initial targets to capture complete or near-complete stratigraphic coverage through continental successions that serve as reference points for western and eastern equatorial Pangaea. © 2020 Copernicus GmbH. All rights reserved

High-throughput, quantitative analyses of genetic interactions in E. coli.

Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli

Characterization of transgene expression and pDNA distribution of the suctioned kidney in mice

We have previously developed an efficient and safe transfection method for the kidney in mice: renal suction-mediated transfection. In this study, we verified the detailed characteristics of transgene expression and plasmid DNA (pDNA) in mice to develop therapeutic strategies and application to gene function analysis in the kidney. After naked pDNA was administered intravenously, the right kidney was immediately suctioned by a tissue suction device. We examined the spatial distribution of transgene expression and pDNA in the suctioned kidney using tissue clearing by CUBIC, ClearT2, and Scale SQ reagents. Spatial distribution analysis showed that pDNA was transfected into extravascular cells and sufficiently delivered to the deep renal cortex. In addition, we revealed that transgene expression occurred mainly in peritubular fibroblasts of the suctioned kidney by tissue clearing and immunohistochemistry. Next, we confirmed the periods of pDNA uptake and activation of transcription factors nuclear factor-κB and activator protein 1 by luciferase assays. Moreover, the use of a pCpG-free plasmid enabled sustained transgene expression in the suctioned kidney. In conclusion, analyses of the spatial distribution and immunostaining of the section suggest that pDNA and transgene expression occurs mainly in peritubular fibroblasts of the suctioned kidney. In addition, we clarified some factors for efficient and/or sustained transgene expression in the suctioned kidney

The ALMA Survey of 70 μm Dark High-mass Clumps in Early Stages (ASHES). I. Pilot Survey: Clump Fragmentation

\ua9 2019. The American Astronomical Society. All rights reserved. The ALMA Survey of 70 μm dark High-mass clumps in Early Stages (ASHES) is designed to systematically characterize the earliest stages and constrain theories of high-mass star formation. Twelve massive (>500 M⊙ ), cold (≤15 K), 3.6-70 μm dark prestellar clump candidates, embedded in infrared dark clouds, were carefully selected in the pilot survey to be observed with the Atacama Large Millimeter/submillimeter Array (ALMA). We have mosaicked each clump (∼1 arcmin2) in continuum and line emission with the 12 m, 7 m, and Total Power (TP) arrays at 224 GHz (1.34 mm), resulting in ∼1.″2 resolution (∼4800 au, at the average source distance). As the first paper in the series, we concentrate on the continuum emission to reveal clump fragmentation. We detect 294 cores, from which 84 (29%) are categorized as protostellar based on outflow activity or "warm core" line emission. The remaining 210 (71%) are considered prestellar core candidates. The number of detected cores is independent of the mass sensitivity range of the observations and, on average, more massive clumps tend to form more cores. We find a large population of low-mass (30 M⊙) prestellar cores (maximum mass 11 M⊙). From the prestellar core mass function, we derive a power-law index of 1.17 \ub1 0.10, which is slightly shallower than Salpeter. We used the minimum spanning tree (MST) technique to characterize the separation between cores and their spatial distribution, and to derive mass segregation ratios. While there is a range of core masses and separations detected in the sample, the mean separation and mass per clump are well explained by thermal Jeans fragmentation and are inconsistent with turbulent Jeans fragmentation. Core spatial distribution is well described by hierarchical subclustering rather than centrally peaked clustering. There is no conclusive evidence of mass segregation. We test several theoretical conditions and conclude that overall, competitive accretion and global hierarchical collapse scenarios are favored over the turbulent core accretion scenario