Is 30-Day Mortality after Admission for Heart Failure an Appropriate Metric for Quality?

Background

The Centers for Medicare and Medicaid Services (CMS) model for publicly reporting national 30-day-risk-adjusted mortality rates for patients admitted with heart failure fails to include clinical variables known to impact total mortality or take into consideration the culture of end-of-life care. We sought to determine if those variables were related to the 30-day mortality of heart failure patients at Geisinger Medical Center.

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.Complexities of natural biological systems make it difficult to understand and define precisely the roles of individual genes and their integrated functions. The use of model organisms with a relatively small number of genes enables the isolation of core biological processes from their complex regulatory networks for extensive characterization. However, even the simplest natural microbes contain many genes of unknown function, as well as genes that can be singly or simultaneously deleted without any noticeable effect on growth rate in a laboratory setting (Hutchison et al. 1999; Glass et al. 2006; Posfai et al. 2006). Ill-defined genes and those mediating functional redundancies both compound the challenge of understanding even the simplest life forms.Toward generating a minimal cell where every gene is essential for the axenic viability of the organism, we are pursuing strategies to reduce the 1-Mb genome of Mycoplasma mycoides JCVI-syn1.0 (Gibson et al. 2010). Because we can (1) introduce this genome into yeast and maintain it as a plasmid (Benders et al. 2010; Karas et al. 2013a); and (2) “transplant” the genome from yeast into mycoplasma recipient cells (Lartigue et al. 2009), genetic tools in yeast are available for reducing this bacterial genome. Several systems offer advanced tools for bacterial genome engineering. Here we further exploit distinctive features of yeast for this purpose.Methods for serially replacing genomic regions with selectable markers are limited by the number of available markers. One effective approach is to reuse the same marker after precise and scarless marker excision (Storici et al. 2001). We have previously used a self-excising marker (Noskov et al. 2010) six times in yeast to generate a JCVI-syn1.0 genome lacking all six restriction systems (JCVI-syn1.0 ∆1-6) (Karas et al. 2013a). Despite the advantages of scarless engineering, sequential procedures are time-consuming. When applied to poorly characterized genes with the potential to interact with other genes, some paths for multigene knockout may lead to dead ends that result from synergistic mutant phenotypes. When a dead end is reached, sequentially returning to a previous genome in an effort to find a detour to a viable higher-order multimutant may be prohibitively time-consuming.An alternative approach to multigene engineering, available in yeast, is to prepare a set of single mutants and combine the deletions into a single strain via cycles of mating and meiotic recombination (Fig. 1A; Pinel et al. 2011; Suzuki et al. 2011, 2012). With a green fluorescent protein (GFP) reporter gene inserted in each deletion locus, the enrichment of higher-order yeast deletion strains in the meiotic population can be accomplished using flow cytometry. Here we apply this method to the JCVI-syn1.0 ∆1-6 exogenous, bacterial genome harbored in yeast to nonsequentially assemble deletions for genes predicted to be individually deletable based on biological knowledge or transposon-mediated disruption data. The functional identification of simultaneously deletable regions is expected to accelerate the effort to construct a minimal genome.Open in a separate windowFigure 1.Progressive clustering of deleted genomic segments. (A) Scheme of the method. Light blue oval represents a bacterial cell. Black ring or horizontal line denotes a bacterial genome, with the orange box indicating the yeast vector used as a site for linearization and recircularization. Gray shape denotes a yeast cell. Green dot in the genome indicates a deletion replaced with a GFP marker. (B) Map of deleted regions. Orange box indicates the yeast vector sequence used for genome linearization and recircularization. Green boxes indicate regions deleted in multimutant mycoplasma strains. Blue boxes denote restriction modification (RM) systems that are also deleted in the strains. (C) Pulsed-gel electrophoresis result for deleted genomes. The starting strain was the JCVI-syn1.0 ∆1–6 strain (1062 kb). Two strains were analyzed for each design of simultaneous deletion (962 kb for eight-deletion or 974 kb for seven-deletion genome). Ladder is a set of yeast chromosomes (New England BioLabs). (D) GFP-RFP ratio sorting result. Standard sorting was compared with sorting based on a GFP-RFP ratio (Methods).     

Quantitative Ethnobotanical Approach to Analyze Local Importance of Tree Species in North Western Himalaya: A Case Study of Ponda Watershed,J&K

The present study has been carried out in the Ponda watershed of district Rajouri (J&K), northwestern Himalaya to analyze local importance of tree species by quantitative ethnobotanical approach and their status of availability in the study area. Data was analyzed using relative frequency of citation (RFC) and use value (UV) index along with Pearson and Spearman correlation coefficients. A total of 34 tree species were encountered represented by 31 genera and 22 families. All identified tree species were classified into 10 general use categories. As per indigenous use, 27 tree species are exploited for firewood followed by 15 for fodder, 12 each for fruits and making agriculture tools whereas very few tree species are being utilized by local people for various other uses. The UV and RFC of different tree species ranged from 0.02 to 0.12 and 0.04 to 0.80, respectively. The Pearson correlation coefficient was found to be higher (0.60) than the Spearman’s rank correlation (0.54), which reflects high linear relationship as compared to monotonic relationship between RFC and UV. The present study showed that Pinus roxburghii was an abundant species, whereas 3 tree species, i.e. Juglans regia, Ficus religiosa and Ulmus wallichiana were observed to be rare. However, according to the IUCN conservation status of the various trees observed in the study area, Juglans regia and Ulmus wallichiana are near threatened and vulnerable species, respectively, which are also exploited for their multiple uses by the locals.     

Kyasanur Forest Disease Outbreak and Vaccination Strategy,Shimoga District,India, 2013–2014

We investigated a Kyasanur Forest disease outbreak in Karnataka, India during December 2013–April 2014. Surveillance and retrospective study indicated low vaccine coverage, low vaccine effectiveness, and spread of disease to areas beyond those selected for vaccination and to age groups not targeted for vaccination. To control disease, vaccination strategies need to be reviewed.