Description: Programmed cell death in Caenorhabditis elegans requires activation of the caspase CED-3, which strictly depends on CED-4. CED-4 forms an octameric apoptosome, which binds the CED-3 zymogen and facilitates its autocatalytic maturation. Despite recent advances, major questions remain unanswered. Importantly, how CED-4 recognizes CED-3 and how such binding facilitates CED-3 activation remain completely unknown. Here we demonstrate that the L2' loop of CED-3 directly binds CED-4 and plays a major role in the formation of an active CED-4–CED-3 holoenzyme. The crystal structure of the CED-4 apoptosome bound to the L2' loop fragment of CED-3, determined at 3.2 Å resolution, reveals specific interactions between a stretch of five hydrophobic amino acids from CED-3 and a shallow surface pocket within the hutch of the funnel-shaped CED-4 apoptosome. Structure-guided biochemical analysis confirms the functional importance of the observed CED-4–CED-3 interface. Structural analysis together with published evidence strongly suggest a working model in which two molecules of CED-3 zymogen, through specific recognition, are forced into the hutch of the CED-4 apoptosome, consequently undergoing dimerization and autocatalytic maturation. The mechanism of CED-3 activation represents a major revision of the prevailing model for initiator caspase activation.

Description: Background Hyponatremia predicts poor prognosis in patients with acute heart failure (AHF). However, the association of the severity of hyponatremia and changes of serum sodium levels with long-term outcome has not been delineated. Methods and Results The study population was drawn from the HARVEST registry ( H eart F a ilure R egistry of Taipei Ve teran s General Hospi t al), so that patients hospitalized for acute heart failure (AHF) composed this study. The National Death Registry was linked to identify the clinical outcomes of all-cause mortality and cardiovascular death, with a follow-up duration of up to 4 years. Among a total of 2556 patients (76.4 years of age, 67% men), 360 had on-admission hyponatremia, defined as a serum sodium level of 〈135 mEq/L on the first day of hospitalization. On-admission hyponatremia was a predictor for all-cause mortality (hazard ratio and 95% CI: 1.43, 1.11–1.83) and cardiovascular mortality (1.50, 1.04–2.17), independent of age, sex, hematocrit, estimated glomerular filtration rate, left ventricular ejection fraction, and prescribed medications. Subjects with severe hyponatremia (〈125 mEq/L) would even have worse clinical outcomes. During hospitalization, a drop of sodium levels of 〉3 mEq/L was associated with a marked increase of mortality than those with minimal or no drop of sodium levels. In addition, subjects with on-admission hyponatremia and drops of serum sodium levels during hospitalization had an incremental risk of death (2.26, 1.36–3.74), relative to those with normonatremia at admission and no treatment-related drop of serum sodium level in the fully adjusted model. Conclusions On-admission hyponatremia is an independent predictor for long-term outcomes in patients hospitalized for AHF. Combined the on-admission hyponatremia with drops of serum sodium levels during hospitalization may make a better risk assessment in AHF patients.

Description: Proatherogenic, hyperlipidemic states demonstrate increases in circulating ligands for scavenger receptor CD36 (eg, oxidized low-density lipoprotein [oxLDL]) and the Na/K-ATPase (eg, cardiotonic steroids). These factors increase inflammation, oxidative stress, and progression of chronic kidney disease. We hypothesized that diet-induced obesity and hyperlipidemia potentiate a CD36/Na/K-ATPase–dependent inflammatory paracrine loop between proximal tubule cells (PTCs) and their associated macrophages and thereby facilitate development of chronic inflammation and tubulointerstitial fibrosis. ApoE –/– and apoE –/– /cd36 –/– mice were fed a high-fat diet for ≤32 weeks and examined for physiologic and histologic changes in renal function. Compared with apoE –/– , apoE –/– /cd36 –/– mice had improved creatinine clearance and blood pressure which corresponded histologically with less glomerular and tubulointerstitial macrophage accumulation, foam cell formation, oxidant stress, and interstitial fibrosis. Coimmunopreciptation and a cell surface fluorescence-based crosslinking assay showed that CD36 and Na/K-ATPase α-1 colocalized in PTCs and macrophages, and this association was increased by oxLDL or the cardiotonic steroid ouabain. OxLDL and ouabain also increased activation of Src and Lyn in PTCs. Cell-free conditioned medium from PTCs treated with oxLDL or ouabain increased macrophage migration. OxLDL, ouabain, or plasma isolated from high-fat diet–fed mice stimulated reactive oxygen species production in PTCs, which was inhibited by N -acetyl-cysteine, apocynin, or Na/K-ATPase α-1 knockdown. These data suggest that ligands generated in hyperlipidemic states activate CD36 and the Na/K-ATPase and potentiate an inflammatory signaling loop involving PTCs and their associated macrophages, which facilitates the development of chronic inflammation, oxidant stress, and fibrosis underlying the renal dysfunction common to proatherogenic, hyperlipidemic states.

Description: The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed toward larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting that their nonrandom distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia , and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome-wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes, and analysis and visualization tools are publicly available.

Description: Accidental or deliberate ionizing radiation exposure can be fatal due to widespread hematopoietic destruction. However, little is known about either the course of injury or the molecular pathways that regulate the subsequent regenerative response. Here we show that the Wnt signaling pathway is critically important for regeneration after radiation-induced injury. Using Wnt reporter mice, we show that radiation triggers activation of Wnt signaling in hematopoietic stem and progenitor cells. β-Catenin-deficient mice, which lack the ability to activate canonical Wnt signaling, exhibited impaired hematopoietic stem cell regeneration and bone marrow recovery after radiation. We found that, as part of the mechanism, hematopoietic stem cells lacking β-catenin fail to suppress the generation of reactive oxygen species and cannot resolve DNA double-strand breaks after radiation. Consistent with the impaired response to radiation, β-catenin-deficient mice are also unable to recover effectively after chemotherapy. Collectively, these data indicate that regenerative responses to distinct hematopoietic injuries share a genetic dependence on β-catenin and raise the possibility that modulation of Wnt signaling may be a path to improving bone marrow recovery after damage.

Description: Background and Purpose— Current literature provides mixed evidence on disparities by race/ethnicity and socioeconomic status in discharge outcomes after hospitalization for acute ischemic stroke. Using comprehensive data from 8 states, we sought to compare inpatient mortality and length of stay by race/ethnicity and socioeconomic status. Methods— We examined all 2007 hospitalizations for acute ischemic stroke in all nonfederal acute care hospitals in Arizona, California, Florida, Maine, New Jersey, New York, Pennsylvania, and Texas. Population was stratified by race/ethnicity (non-Hispanic whites, non-Hispanic blacks, and Hispanics) and socioeconomic status, measured by median income of patient zip code. For each stratum, we estimated risk-adjusted rates of inpatient mortality and longer length of stay (greater than median length of stay). We also compared the hospitals where these subpopulations received care. Results— Hispanic and black patients accounted for 14% and 12% of all ischemic stroke admissions (N=147 780), respectively, and had lower crude inpatient mortality rates (Hispanic=4.5%, blacks=4.4%; all P 〈0.001) compared with white patients (5.8%). Hispanic and black patients were younger and fewer had any form of atrial fibrillation. Adjusted for patient risk, inpatient mortality was similar by race/ethnicity, but was significantly higher for low-income area patients than that for high-income area patients (odds ratio, 1.08; 95% confidence interval, 1.02–1.15). Risk-adjusted rates of longer length of stay were higher among minority and low-income area populations. Conclusions— Risk-adjusted inpatient mortality was similar among patients by race/ethnicity but higher among patients from lower income areas. However, this pattern was not evident in sensitivity analyses, including the use of mechanical ventilation as a partial surrogate for stroke severity.

Description: Spatial organization of different epigenomic marks was used to infer functions of the epigenome. It remains unclear what can be learned from the temporal changes of the epigenome. Here, we developed a probabilistic model to cluster genomic sequences based on the similarity of temporal changes of multiple epigenomic marks during a cellular differentiation process. We differentiated mouse embryonic stem (ES) cells into mesendoderm cells. At three time points during this differentiation process, we used high-throughput sequencing to measure seven histone modifications and variants—H3K4me1/2/3, H3K27ac, H3K27me3, H3K36me3, and H2A.Z; two DNA modifications—5-mC and 5-hmC; and transcribed mRNAs and noncoding RNAs (ncRNAs). Genomic sequences were clustered based on the spatiotemporal epigenomic information. These clusters not only clearly distinguished gene bodies, promoters, and enhancers, but also were predictive of bidirectional promoters, miRNA promoters, and piRNAs. This suggests specific epigenomic patterns exist on piRNA genes much earlier than germ cell development. Temporal changes of H3K4me2, unmethylated CpG, and H2A.Z were predictive of 5-hmC changes, suggesting unmethylated CpG and H3K4me2 as potential upstream signals guiding TETs to specific sequences. Several rules on combinatorial epigenomic changes and their effects on mRNA expression and ncRNA expression were derived, including a simple rule governing the relationship between 5-hmC and gene expression levels. A Sox17 enhancer containing a FOXA2 binding site and a Foxa2 enhancer containing a SOX17 binding site were identified, suggesting a positive feedback loop between the two mesendoderm transcription factors. These data illustrate the power of using epigenome dynamics to investigate regulatory functions.

Description: The draft genome of the pear ( Pyrus bretschneideri ) using a combination of BAC-by-BAC and next-generation sequencing is reported. A 512.0-Mb sequence corresponding to 97.1% of the estimated genome size of this highly heterozygous species is assembled with 194 x coverage. High-density genetic maps comprising 2005 SNP markers anchored 75.5% of the sequence to all 17 chromosomes. The pear genome encodes 42,812 protein-coding genes, and of these, ~28.5% encode multiple isoforms. Repetitive sequences of 271.9 Mb in length, accounting for 53.1% of the pear genome, are identified. Simulation of eudicots to the ancestor of Rosaceae has reconstructed nine ancestral chromosomes. Pear and apple diverged from each other ~5.4–21.5 million years ago, and a recent whole-genome duplication (WGD) event must have occurred 30–45 MYA prior to their divergence, but following divergence from strawberry. When compared with the apple genome sequence, size differences between the apple and pear genomes are confirmed mainly due to the presence of repetitive sequences predominantly contributed by transposable elements (TEs), while genic regions are similar in both species. Genes critical for self-incompatibility, lignified stone cells (a unique feature of pear fruit), sorbitol metabolism, and volatile compounds of fruit have also been identified. Multiple candidate SFB genes appear as tandem repeats in the S -locus region of pear; while lignin synthesis-related gene family expansion and highly expressed gene families of HCT , C3'H , and CCOMT contribute to high accumulation of both G-lignin and S-lignin. Moreover, alpha-linolenic acid metabolism is a key pathway for aroma in pear fruit.

Description: Rationale: Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ, such as the heart, especially those exhibiting adult phenotypes on depletion. Objective: We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes. Methods and Results: The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an insertional mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of 1 embryonic lethal, 1 larval lethal, and 1 adult recessive mutant exhibiting cardiac hypertrophy at 1 year of age. Further characterization of these mutants uncovered an essential function of methionine adenosyltransferase II, α a (mat2aa) in cardiogenesis, an essential function of mitochondrial ribosomal protein S18B (mrps18b) in cardiac mitochondrial homeostasis, as well as a function of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) in adult cardiac hypertrophy. Conclusions: We demonstrate that transposon-based gene trapping is an efficient approach for identifying both embryonic and adult recessive mutants with cardiac expression. The generation of a zebrafish insertional cardiac mutant collection shall facilitate the annotation of a vertebrate cardiac genome, as well as enable heart-based adult screens.

Description: Objective— S-Adenosylhomocysteine (SAH) is a better predictor of cardiovascular disease than homocysteine is, and it has been implicated in mediating the pathogenicity of hyperhomocysteinemia in atherosclerosis via an epigenetic mechanism. However, the underlying mechanism remains unclear. Here, we tested the hypothesis whether the effect of SAH on atherosclerosis is involved in epigenetic regulation of endoplasmic reticulum stress. Approach and Results— A total of 48 apolipoprotein E–deficient mice at 8 weeks were randomly divided into 4 groups (n=12 for each group). The control group was fed a conventional diet, the adenosine dialdehyde group was fed a diet that was supplemented with the SAH hydrolase inhibitor adenosine dialdehyde, and the other 2 groups were intravenously injected with a retrovirus that expressed either SAH hydrolase short hairpin RNA or scrambled short hairpin RNA semiweekly for 16 weeks. Plasma SAH levels and atherosclerotic lesion size were significantly increased in adenosine dialdehyde and SAH hydrolase short hairpin RNA groups when compared with control group. Expression of endoplasmic reticulum stress markers glucose-regulated protein-78 and CEBP-homologous protein was significantly increased in the mice with elevated plasma SAH levels. Moreover, plasma SAH was negatively associated with a decrease in the expression of trimethylated histone H3 lysine 9 and histone methyltransferases. Chromatin immunoprecipitation assays showed a significant decrease in trimethylated histone H3 lysine 9 occupancy at the glucose-regulated protein-78 and CEBP-homologous protein promoters in mice treated with adenosine dialdehyde and SAH hydrolase short hairpin RNA when compared with control mice. Conclusions— Our results suggest that elevated plasma SAH levels–accelerated atherosclerosis was associated with the activation of endoplasmic reticulum stress via modulation of histone methylation.