Use of the SAMe-TT2R2 Score to Predict Good Anticoagulation Control with Warfarin in Chinese Patients with Atrial Fibrillation: Relationship to Ischemic Stroke Incidence

Background

The efficacy and safety of warfarin therapy for stroke prevention in atrial fibrillation (AF) depends on the time in therapeutic range (TTR). We aimed to assess the predictive ability of SAMe-TT₂R₂ score in Chinese AF patients on warfarin, whose TTR is notoriously poor.

Methods and Results

This is a single-centre retrospective study. Patients with non-valvular AF on warfarin diagnosed between 1997 and 2011 were stratified according to SAMe-TT₂R₂ score, and TTR was calculated using Rosendaal method. The predictive power of SAMe-TT₂R₂ scores for good TTR i.e. >70% was assessed. We included 1,428 Chinese patients (mean age 76.2±8.7 years, 47.5% male) with non-valvular AF on warfarin. The mean and median TTR were 38.2±24.4% and 38.8% (interquartile range: 17.9% and 56.2%) respectively. TTR decreased progressively with increasing SAMe-TT₂R₂ score (p = 0.016). When the cut-off value of SAMe-TT₂R₂ score was set to 2, the sensitivity and specificity to predict TTR<70% were 85.7% and 17.8%, respectively. The corresponding positive and negative predictive values were 10.1% and 92.0%. After a mean follow-up of 4.7±3.6 years, 338 patients developed an ischemic stroke (4.96%/year). Patients with TTR≥70% had a lower annual risk of ischemic stroke of 3.67%/year compared with than those with TTR<70% (5.13%/year)(p = 0.08). Patients with SAMe-TT₂R₂ score ≤2 had the lowest risk of annual risk of ischemic stroke (3.49%/year) compared with those with SAMe-TT₂R₂ score = 3 (4.56%/year), and those with SAMe-TT₂R₂ score ≥4 (6.41%/year)(p<0.001). There was also a non-significant trend towards more intracranial hemorrhage with increasing SAMe-TT₂R₂ score.

Conclusions

The SAMe-TT₂R₂ score correlates well with TTR in Chinese AF patients, with a score >2 having high sensitivity and negative predictive values for poor TTR. Ischemic stroke risk increased progressively with increasing SAMe-TT₂R₂ score, consistent with poorer TTRs at high SAMe-TT₂R₂ scores.     

Antihypertensive effect of mechanism-based inhibition of renal arachidonic acid omega-hydroxylase activity

The cytochrome P-450 eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor that is implicated in the regulation of blood pressure. The identification of selective inhibitors of renal 20-HETE formation for use in vivo would facilitate studies to determine the systemic effects of this eicosanoid. We characterized the acetylenic fatty acid sodium 10-undecynyl sulfate (10-SUYS) as a potent and selective mechanism-based inhibitor of renal 20-HETE formation. A single dose of 10-SUYS caused an acute reduction in mean arterial blood pressure in 8-wk-old spontaneously hypertensive rats. The decrease in mean arterial pressure was maximal 6 h after 10-SUYS treatment (17.9 +/- 3.2 mmHg; P < 0.05), and blood pressure returned to baseline levels within 24 h after treatment. Treatment with 10-SUYS was associated with a decrease in urinary 20-HETE formation in vivo and attenuation of the vasoconstrictor response of renal interlobar arteries to ANG II in vitro. These results provide further evidence that 20-HETE plays an important role in the regulation of blood pressure in the spontaneously hypertensive rat.     

Affinity selection of DNA-binding proteins from yeast genomic DNA libraries by improved lambda phage display vector

Phage display is a useful means of identifying and selecting proteins of interest that bind specific targets. In order to examine the potential of phage display for the genome-wide screening of DNA-binding proteins, we constructed yeast genomic libraries using lambda foo-based vectors devised in this work. After affinity selection using GAL4 UAS(G) as a probe, phages expressing GAL4 were enriched approximately 5 x 10(5)-fold from the library. Approximately 90% of polypeptides encoded in correct translation reading frames by the selected phages were known or putative polynucleotide-binding proteins. This result clearly indicates that the modified lambda phage display vector in combination with our enrichment technique has great potential for the enrichment of DNA-binding proteins in a sequence-specific manner.     

Validation of single nucleotide polymorphism quantification in pooled DNA samples with SNaPIT. A glycosylase-mediated methods for polymorphism detection method

Association studies using genome scans to identify quantitative trait loci for multifactorial disorders, with anything approaching reasonable power, have been compromised by the need for a very dense array of genetic markers and large numbers of affected individuals. These requirements impose enormous burdens on the genotyping capacity for most laboratories. DNA pooling has been proposed as a possible approach to reduce genotyping costs and effort. We report on the application of the SNaPIT™ technology to evaluate allele frequencies in pooled DNA samples and conclude that it offers a cost effective, efficient and accurate estimator and provides several advantages over competing technologies in this regard.     

Role of Ndt80, Sum1, and Swe1 as targets of the meiotic recombination checkpoint that control exit from pachytene and spore formation in Saccharomyces cerevisiae

The meiotic recombination checkpoint, which is triggered by defects in recombination or chromosome synapsis, arrests sporulating cells of Saccharomyces cerevisiae at pachytene by preventing accumulation of active Clb-Cdc28. We compared the effects of manipulating the three known targets of the meiotic recombination checkpoint, NDT80, SWE1, and SUM1, in dmc1-arrested cells. Ndt80 is an activator of a set of middle sporulation-specific genes (MSGs), which includes CLB genes and genes involved in spore wall formation; Swe1 inhibits Clb-Cdc28 activity; and Sum1 is a repressor of NDT80 and some MSGs. Activation of the checkpoint leads to inhibition of Ndt80 activity and to stabilization of Swe1 and Sum1. Thus, dmc1-arrested cells fail to express MSGs, arrest at pachytene, and do not form spores. Our study shows that dmc1/dmc1 sum1/sum1 cells expressed MSGs prematurely and at high levels, entered the meiotic divisions efficiently, and in some cases formed asci containing mature spores. In contrast, dmc1/dmc1 swe1/swe1 cells expressed MSGs at a very low level, were inefficient and delayed in entry into the meiotic divisions, and never formed mature spores. We found that cells of dmc1/dmc1 sum1/sum1 ndt80/ndt80 and dmc1/dmc1 swe1/swe1 ndt80/ndt80 strains arrested at pachytene and that dmc1/dmc1 or dmc1/dmc1 swe1/swe1 cells overexpressing NDT80 were less efficient in bypassing checkpoint-mediated arrest than dmc1/dmc1 sum1/sum1 cells. Our results are consistent with previous suggestions that increased Clb-Cdc28 activity, caused by mutation of SWE1 or by an NDT80-dependent increase in CLB expression, allows dmc1/dmc1 cells to exit pachytene and that subsequent upregulation of Ndt80 activity by a feedback mechanism promotes entry into the meiotic divisions. Spore morphogenesis, however, requires efficient and timely activation of MSGs, which we speculate was achieved in dmc1/dmc1 sum1/sum1 cells by premature expression of NDT80.     

Karyotypes of metaphase chromosomes in diploid populations of Dendranthema zawadskii and related species (Asteraceae) from Korea: diversity and evolutionary implications

Although the Dendranthema zawadskii complex has been known to comprise a series of polyploids (4x, 6x, 8x), we found diploid individuals (with 2 n=18) to occur in four populations of D. zawadskii var. latilobum in the southern region of Korea. Karyotypes of metaphase chromosomes were diverse because numbers of metacentric, submetacentric, and acrocentric chromosomes differ even within a population. A total of 17 karyotypes were found in 31 diploid individuals collected from the four populations. The karyotypes were also diverse in the presence or absence of chromosomes with a secondary constriction on a short or long arm and, if present, in the number of such chromosomes. They were further diverse in the presence or absence of non-homologous chromosome(s), the presence or absence of a chromosome with a satellite, and, if present, how many and where satellites are present. Almost the same pattern of diversity was found in diploid individuals (with 2 n=18) of D. boreale and D. indicum as well, irrespective of whether they occur together with D. zawadskii var. latilobum or not. Structural features of chromosomes in the variously different karyotypes suggest that reciprocal translocation and the hybridization between individuals with different karyotypes had repeatedly occurred not only in D. zawadskii var. latilobum, but also in D. boreale and D. indicum. Morphologically intermediate individuals between D. zawadskii var. latilobum and D. indicum suggests that the hybridization occur with different species as well.     

The calpain small subunit gene is essential: its inactivation results in embryonic lethality

Creation of transgenic (knockout) mice deficient in calpain small (30 kDa) subunit gene was undertaken to clarify the proposed role of the small subunit for calpain proteolytic activity and to gain insight into the importance of the gene in the whole animal. The gene was targeted and disrupted in embryonic stem cells by homologous recombination, and chimeric mice were generated. Heterozygous F1 generation mice were crossed to obtain F2 generation. Among F2 generation mice, we found only wild-type and heterozygous animals in the 80 pups genotyped to date; no homozygous mice have been found, although 20 were expected. The heterozygotes had no apparent phenotypic abnormalities. Analysis of their tissues revealed no significant difference in mRNA expression, protein content, or proteolytic activity in comparison with their wild-type littermates. Genotyping of fetuses at different stages of development also revealed only wild-type and normal heterozygous fetuses. No moribund embryos or resorption sites were observed in the uterine cavity. The results indicate that at least one normal allele is essential for postnatal survival. Disruption of both alleles appears to be lethal in very early fetal development.     

Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.     

Light-induced pigment granule migration in the retinular cells of Drosophila melanogaster. Comparison of wild type with ERG-defective mutants

The dependence of pigment granule migration (PGM) upon the receptor potential was examined using several strains of electroretinogram (ERG)- defective mutants of Drosophila melanogaster. The mutants that have a defective lamina component but a normal receptor component of the ERG (no on-transient A [nonA] and tan) exhibited normal pigment granule migration. The mutants that have very small or no receptor potentials (certain no receptor potential A [norpA] alleles), on the other hand, exhibited no PGM. In the case of the temperature-sensitive norpA mutant, norpAH52, normal PGM was present at 17 degrees but not at 32 degrees C or above, corresponding to its electrophysiological phenotype. In the transient receptor potential (trp) mutant, whose receptor potential decays to the baseline within a few seconds during a sustained light stimulus, the pigment granules initially moved close to the rhabdomere when light was turned on but moved away after about 5 s during a sustained light stimulus. All these results lend strong support to the notion that PGM is initiated by a light-evoked depolarization of the receptor membrane, i.e., the receptor potential. However, under certain experimental conditions, the receptor potentials failed to induce PGM in the trp mutant. The depolarization of the receptor, thus, appears to be closely associated with PGM but is not a sufficient condition for PGM.