Impact of diabetes mellitus on acute outcomes of percutaneous coronary intervention in chronic total occlusions: insights from a US multicentre registry.

AIM: To examine the impact of diabetes mellitus on procedural outcomes of patients who underwent percutaneous coronary intervention for chronic total occlusion. METHODS: We assessed the impact of diabetes mellitus on the outcomes of percutaneous coronary intervention for chronic total occlusion among 1308 people who underwent such procedures at 11 US centres between 2012 and 2015. RESULTS: The participants' mean ± sd age was 66 ± 10 years, 84% of the participants were men and 44.6% had diabetes. As compared with participants without diabetes, participants with diabetes were more likely to have undergone coronary artery bypass graft surgery (38 vs 31%; P = 0.006), and to have had previous heart failure (35 vs 22%; P = 0.0001) and peripheral arterial disease (19 vs 13%; P = 0.002). They also had a higher BMI (31 ± 6 kg/m2 vs 29 ± 6 kg/m2 ; P = 0.001), similar Japanese chronic total occlusion scores (2.6 ± 1.2 vs 2.5 ± 1.2; P = 0.82) and similar final successful crossing technique: antegrade wire escalation (46 vs 47%; P = 0.66), retrograde (30 vs 28%; P = 0.66) and antegrade dissection re-entry (24 vs 25%; P = 0.66). Technical (91 vs 90%; P = 0.80) and procedural (89 vs 89%; P = 0.93) success was similar in the two groups, as was the incidence of major adverse cardiac events (2.2 vs 2.5%; P = 0.61). CONCLUSIONS: In a contemporary cohort of people undergoing percutaneous coronary intervention for chronic total occlusion, nearly one in two (45%) had diabetes mellitus. Procedural success and complication rates were similar in people with and without diabetes.

Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map.

Single nucleotide polymorphisms (SNPs) are valuable genetic markers of human disease. They also comprise the highest potential density marker set available for mapping experimentally derived mutations in model organisms such as Caenorhabditis elegans. To facilitate the positional cloning of mutations we have identified polymorphisms in CB4856, an isolate from a Hawaiian island that shows a uniformly high density of polymorphisms compared with the reference Bristol N2 strain. Based on 5.4 Mbp of aligned sequences, we predicted 6,222 polymorphisms. Furthermore, 3,457 of these markers modify restriction enzyme recognition sites ('snip-SNPs') and are therefore easily detected as RFLPs. Of these, 493 were experimentally confirmed by restriction digest to produce a snip-SNP map of the worm genome. A mapping strategy using snip-SNPs and bulked segregant analysis (BSA) is outlined. CB4856 is crossed into a mutant strain, and exclusion of CB4856 alleles of a subset of snip-SNPs in mutant progeny is assessed with BSA. The proximity of a linked marker to the mutation is estimated by the relative proportion of each form of the biallelic marker in populations of wildtype and mutant genomes. The usefulness of this approach is illustrated by the rapid mapping of the dyf-5 gene.

Single-nucleotide polymorphisms in the public domain: how useful are they?

There is a concerted effort by a number of public and private groups to identify a large set of human single-nucleotide polymorphisms (SNPs). As of March 2001, 2.84 million SNPs have been deposited in the public database, dbSNP, at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/SNP/). The 2.84 million SNPs can be grouped into 1.65 million non-redundant SNPs. As part of the International SNP Map Working Group, we recently published a high-density SNP map of the human genome consisting of 1.42 million SNPs (ref. 3). In addition, numerous SNPs are maintained in proprietary databases. Our survey of more than 1,200 SNPs indicates that more than 80% of TSC and Washington University candidate SNPs are polymorphic and that approximately 50% of the candidate SNPs from these two sources are common SNPs (with minor allele frequency of > or =20%) in any given population.

A general approach to single-nucleotide polymorphism discovery.

Single-nucleotide polymorphisms (SNPs) are the most abundant form of human genetic variation and a resource for mapping complex genetic traits. The large volume of data produced by high-throughput sequencing projects is a rich and largely untapped source of SNPs (refs 2, 3, 4, 5). We present here a unified approach to the discovery of variations in genetic sequence data of arbitrary DNA sources. We propose to use the rapidly emerging genomic sequence as a template on which to layer often unmapped, fragmentary sequence data and to use base quality values to discern true allelic variations from sequencing errors. By taking advantage of the genomic sequence we are able to use simpler yet more accurate methods for sequence organization: fragment clustering, paralogue identification and multiple alignment. We analyse these sequences with a novel, Bayesian inference engine, POLYBAYES, to calculate the probability that a given site is polymorphic. Rigorous treatment of base quality permits completely automated evaluation of the full length of all sequences, without limitations on alignment depth. We demonstrate this approach by accurate SNP predictions in human ESTs aligned to finished and working-draft quality genomic sequences, a data set representative of the typical challenges of sequence-based SNP discovery.

Variations in the Course of the Carotid Arteries in Patients with Retropharyngeal Parathyroid Adenomas.

The carotid arteries, classically described as taking a relatively straight course through the neck, deviate medially in a minority of patients. At the extreme, the internal carotid arteries may "kiss" in the midline, coming extremely close to the pharyngeal wall. In this clinical report, we describe 5 patients with primary hyperparathyroidism, all with ectopic retropharyngeal parathyroid adenomas but all with varying carotid artery anatomy. We describe these variations using a previously developed clinical grading system that highlights 1) the relationship between carotid artery location and risk of injury during pharyngeal procedures and 2) the importance of universal, objective criteria to classify carotid anatomy. Radiologists should be familiar with variations in carotid anatomy and communicate them to the operative team.

A Simple Formula to Estimate Parathyroid Weight on 4D-CT, Predict Pathologic Weight, and Diagnose Parathyroid Adenoma in Patients with Primary Hyperparathyroidism.

BACKGROUND AND PURPOSE: Parathyroid gland weight is a clinically relevant parameter used to diagnose parathyroid adenomas intraoperatively. We evaluated the accuracy of a formula to estimate parathyroid weight on preoperative 4D-CT. MATERIALS AND METHODS: A single-institution retrospective study was performed in patients with primary hyperparathyroidism who underwent 4D-CT between January 2013 and December 2014 with subsequent parathyroidectomy and surgical cure. All patients had correct localization of a solitary parathyroid adenoma. The longest 3 dimensions of all identified parathyroid glands were measured on CT, and weight was estimated using the formula: weight4D-CT (mg) = 1 mg/mm3 × Length (mm) × Width (mm) × Height (mm) × π/6. We correlated weight4D-CT with pathology specimen weight (weightpathology). Using receiver operating characteristic analysis, we estimated the performance of weight4D-CT to discriminate a parathyroid adenoma from normal glands on 4D-CT and determined the optimal threshold based on the Youden index. RESULTS: One hundred sixteen patients (85 women, 31 men) were evaluated. Weight4D-CT was shown to be strongly correlated with weightpathology as demonstrated by Spearman ρ = 0.73 (P < .01), concordance correlation coefficient = 0.92 (95% CI, 0.89-0.94), and Cronbach α = 0.96. The performance of weight4D-CT for the diagnosis of parathyroid adenoma was excellent, with an area under the curve of 0.955 (95% CI, 0.925-0.985; P < .001). Based on the Youden index, the optimal threshold was >50 mg, with a sensitivity of 96.7% and a specificity of 95.7%. CONCLUSIONS: Radiologists can accurately estimate parathyroid adenoma weight on 4D-CT. This metric is highly correlated with pathologic weight, and a threshold cutoff of >50 mg can be used to distinguish parathyroid adenoma from normal glands.

Alpha-particle emissivity of the moon: an observed upper limit.

Measurements made by the moon-orbiting spacecraft Explorer 35 during 1967-1968 show that it is unlikely that the alpha-particle emissivity of the moon is greater than 0.064 per square centimeter per second per steradian and exceedingly unlikely that it is greater than 0.128, these values being respectively 0.1 and 0.2 of the provisional estimates made by Kraner et al. in 1966. This result implies that the abundance of uranium-238 in the outer crust (approximately a few meters thick) of the moon is much less than that typical of the earth's lithosphere, though it is consistent with the abundance of uranium-238 in terrestrial basalt or in chondritic meteorites.

Effects of Bacillus subtilis var. natto and Saccharomyces cerevisiae mixed fermented feed on the enhanced growth performance of broilers.

Bacillus subtilis var. natto N21 (Bac; for greater proteolytic capacity) and Saccharomyces cerevisiae Y10 (Sac; for greater acidic capacity) were applied to produce a 2-stage combined fermentation feed. This study investigated whether the enhancement of Bac+Sac fermented feed on broiler growth performance was due to the probiotics per se or due to the fermentation process. Trial 1 included 1-d-old broiler chicks (n=144) randomly assigned to control, water added (same as in the fermentation feed, 23%), and Bac+Sac fermented feed (FBac+Sac) treatments with 4 replicates. Trial 2 included 21-d-old broiler chickens (n=12) assigned into control and FBac+Sac groups for a metabolic trial for nutrient availability. Trial 3 included 1-d-old male broiler chicks (n=216) randomly assigned into 6 treatments with 3 replicates. Treatments included a control, Sac fermented feed (FSac), FBac+Sac, Bac powder (PBac), Sac powder (PSac), and Bac+Sac powder (PBac+Sac). The results from trial 1 showed that FBac+Sac increased BW and feed intake (P<0.05) in 21- and 39-d-old chickens. The water-added group showed decreased BW, weight gain, and feed intake (P<0.05). Trial 2 showed that FBac+ Sac increased gross energy availability (P<0.05). Trial 3 showed that FBac+Sac increased 21- and 39-d-old BW and weight gain (P<0.05). Diets supplemented with probiotic powder or fermented with Sac did not improve broiler growth performance (P>0.05). The growth performance improvement of the FBac+Sac treatment was probably not due to the added water, probiotic powder inclusion, or through single-strain fermentation, but due to the 2-stage fermentation process using Bac and Sac strains.

Identification of two distinct MYC breakpoint clusters and their association with various IGH breakpoint regions in the t(8;14) translocations in sporadic Burkitt-lymphoma.

The chromosomal translocation t(8;14) is the hallmark of Burkitt's-lymphoma (BL) and fuses the proto-oncogene c-MYC to the IGH locus. We analyzed the genomic structure of MYC/IGH fusions derived from a large series of 78 patients with t(8;14) and asked (i) whether distinct breakpoint clusters exist within the MYC gene and (ii) whether any pairwise association between particular IGH and MYC breakpoints exist. Identification of such associations will help elucidate the etiology of the breaks on the MYC locus. Scan statistic analyses revealed two distinct, but large clusters within c-MYC containing 60/78 (77%) of the breakpoints. Clusters 1 and 2 were 560 and 779 bp in length within a 4555 bp breakpoint cluster region. Breaks within IGH switch mu and joining region did not differ with respect to their corresponding MYC breakpoints. However, there was a highly significant correlation between breakpoints 5' of MYC cluster 1 and fusions to IGH switch gamma region and breakpoints downstream of MYC cluster 2 and fusions to IGH switch alpha region (chi(2)-test: P<0.005). Chromatin changes governing choice of IGH-Fc region recombination may parallel changes in the MYC gene 5' region chromatin leading to some degree of coordinated ontological specificity in breakpoint location.

Comparison of morphological changes of muscle fibers in response to dynamic electrical muscle contraction and dynamic hydraulic stimulation in a rat hindlimb disuse model.

This study attempted to compare the muscle fiber morphological responses to dynamic electrical muscle stimulation (DEMS) and dynamic hydraulic stimulation (DHS) in rats under hindlimb suspension (HLS). DEMS at 1 Hz, 50 Hz and 100 Hz for 10 min/day, 5 days/week were introduced to the animals' right quadriceps. Static and 2 Hz DHS were introduced to the right tibiae of other animal groups on a "10 min on - 5 min off - 10 min on" loading regime for 5 days/week. In the end of the 4-week experiments, histological changes in the corresponding soleus, gastrocnemius and quadriceps of the stimulated sites were examined. Compared to age-matched, HLS led to muscle atrophy and strongly reduced muscle wet weights and averaged cross-sectional fiber areas. Among the tested DEMS frequencies, the averaged cross-sectional quadriceps fiber area in the 50 Hz group was 29 % larger than the 100 Hz group. In contrast, difference in the muscle fiber response to the static and 2 Hz DHS was not observed in either soleus or gastrocnemius. Muscle fiber morphological responses to the active DEMS was in a load frequency dependent manner under disuse condition. Relatively passive compressions, either via static or 2Hz DHS, were unable to induce any difference in the muscle fiber responses under functional disuse.

Identification of a sister gene to P-glycoprotein.

The P-glycoproteins (Pgps) are a small family of transport proteins associated with the multidrug resistance phenotype of cell lines selected for growth in cytotoxic drugs. Utilizing low stringency screening, we have identified a novel gene closely related to the Pgps expressed in the pig and other mammalian liver which we have called Sister of P-glycoprotein (spgp). Sequence of this gene shows it to be a member of the ATP-binding cassette family of transporters and the gene most closely related to Pgp identified to date. The function of spgp is not known, but it can be recognized by at least one Pgp mAb, C219. This cross-reactivity has implications for expression studies in tissues and tumors utilizing this and other Pgp antibodies.

Taxol resistance mediated by transfection of the liver-specific sister gene of P-glycoprotein.

The sister gene of P-glycoprotein (Spgp) is a liver-specific ATP-binding cassette protein highly related to the P-glycoprotein (Pgp) family (S. Childs et al, Cancer Res., 55: 2029-2034, 1995). Spgp appears to be related to the Pgp family by an ancient duplication occurring before the division of fish and mammals. P-Glycoproteins have diverse functions including broad specificity multidrug resistance in cell lines and tumors, detoxification of tissues such as the intestine and blood-brain barrier, and phosphatidylcholine transport in liver. Spgp is a Mr approximately 170,000 glycosylated plasma membrane protein localized to the canalicular surface of hepatocytes in the rat liver. The full-length cDNA of Spgp was isolated from rat, and its expression was characterized in situ and in transfected cells. The expression of Spgp correlates with the differentiation of hepatocytes and is seen only in late liver development. It is not observed in hepatoma cell lines. The physiological function of Spgp in liver is unknown, but it maps to 2q31 in humans, in the vicinity of liver transport disorders for bile acids and cholesterol. Spgp may therefore be involved in some aspect of bile acid or cholesterol metabolism. Spgp transfectants have a low level resistance to Taxol but not to other drugs that form part of the multidrug resistance phenotype. This resistance is reversible by the Pgp-reversing agents cyclosporin A, PSC833, and verapamil, suggesting a conservation in some functions of Pgps across large evolutionary distance.

A new insight into the cellular regulation of aqueous outflow: how trabecular meshwork endothelial cells drive a mechanism that regulates the permeability of Schlemm's canal endothelial cells.

AIM: To test the hypothesis that trabecular meshwork endothelial cells (TMEs) increase the permeability of Schlemm's canal endothelial cells (SCEs) by actively releasing ligands that modulate the barrier properties of SCEs. METHODS: The TMEs were first irradiated with a laser light and allowed to condition the medium, which is then added to SCEs. The treatment response is determined by both measuring SCE permeability (flow meters) and the differential expression of genes (Affymetrix chips and quantitative polymerase chain reaction (PCR)). The cytokines secreted by the treated cells were identified using ELISA and the ability of these cytokines to increase permeability is tested directly after their addition to SCEs in perfusion experiments. RESULTS: SCEs exposed to medium conditioned by the light activated TMEs (TME-cm) respond by undergoing a differential expression (DE) of 1,120 genes relative to controls. This response is intense relative to a DE of only 12 genes in lasered SCEs. The TME-cm treatment of SCEs increased the SCE permeability fourfold. The role of cytokines in these responses is supported by two findings: adding specific cytokines established to be secreted by lasered TMEs to SCEs increases permeability; and inactivating the TME-cm by boiling or diluting, abrogates these conditioned media permeability effects. CONCLUSION: These experiments show that TMEs can regulate SCE permeability and that it is likely that TMEs have a major role in the regulation of aqueous outflow. This novel TME driven cellular mechanism has important implications for the pathogenesis of glaucoma and the mechanism of action of laser trabeculoplasty. Ligands identified as regulating SCE permeability have potential use for glaucoma therapy.

Gene expression of INPP5F as an independent prognostic marker in fludarabine-based therapy of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease. Various disease-related and patient-related factors have been shown to influence the course of the disease. The aim of this study was to identify novel biomarkers of significant clinical relevance. Pretreatment CD19-separated lymphocytes (n=237; discovery set) and peripheral blood mononuclear cells (n=92; validation set) from the REACH trial, a randomized phase III trial in relapsed CLL comparing rituximab plus fludarabine plus cyclophosphamide with fludarabine plus cyclophosphamide alone, underwent gene expression profiling. By using Cox regression survival analysis on the discovery set, we identified inositol polyphosphate-5-phosphatase F (INPP5F) as a prognostic factor for progression-free survival (P<0.001; hazard ratio (HR), 1.63; 95% confidence interval (CI), 1.35-1.98) and overall survival (P<0.001; HR, 1.47; 95% CI, 1.18-1.84), regardless of adjusting for known prognostic factors. These findings were confirmed on the validation set, suggesting that INPP5F may serve as a novel, easy-to-assess future prognostic biomarker for fludarabine-based therapy in CLL.

Effect of cerebral ischemia on calcium/calmodulin-dependent protein kinase II activity and phosphorylation.

The effects of cerebral ischemia on calcium/calmodulin-dependent kinase II (CaM kinase II) were investigated using the rat four-vessel occlusion model. In agreement with previous results using rat or gerbil models of cerebral ischemia or a rabbit model of spinal cord ischemia, this report demonstrates that transient forebrain ischemia leads to a reduction in CaM kinase II activity within 5 min of occlusion onset. Loss of activity from the cytosol fractions of homogenates from the neocortex, striatum, and hippocampus correlated with a decrease in the amount of CaM kinase alpha and beta isoforms detected by immunoblotting. In contrast, there was an apparent increase in the amount of CaM kinase alpha and beta in the particulate fractions. The decrease in the amount of CaM kinase isoforms from the cytosol but not the particulate fractions was confirmed by autophosphorylation of CaM kinase II after denaturation and renaturation in situ of the blotted proteins. These results indicate that ischemia causes a rapid inhibition of CaM kinase II activity and a change in the partitioning of the enzyme between the cytosol and particulate fractions. CaM kinase II is a multifunctional protein kinase, and the loss of activity may play a critical role in initiating the changes leading to ischemia-induced cell death. To identify a structural basis for the decrease in enzyme activity, tryptic peptide maps of CaM kinase II phosphorylated in vitro were compared. Phosphopeptide maps of CaM kinase alpha from particulate fractions of control and ischemic samples revealed not only reduced incorporation of phosphate into the protein but also the absence of a limited number of peptides in the ischemic samples. This suggested that certain sites are inaccessible, possibly due to a conformational change, a covalent modification of CaM kinase II, or steric hindrance by an associated molecule. Verifying one of these possibilities should help to elucidate the mechanism of ischemia-induced modulation of CaM kinase II.

Differential effects of ischemia and reperfusion on c-Jun N-terminal kinase isoform protein and activity.

Activation of the c-Jun N-terminal (JNK) or stress-activated protein kinases (SAPK) is associated with a wide range of disparate cellular responses to extracellular stimuli, including either induction of or protection from apoptosis. This study investigates the effect of ischemia and reperfusion on JNK isoform activities using a reversible rabbit spinal cord ischemia model. High basal JNK activity, attributed to the p46 JNK1 isoform, was expressed in the CNS of untreated rabbits. JNK activity decreased in the lumbar spinal cord of rabbits occluded for 15-60 min. During reperfusion animals occluded for 15 min recovered neurological function and JNK activity returned to normal levels. In contrast animals occluded for 60 min remained permanently paraplegic and JNK activity was half the control activity after 18 h of reperfusion. In these animals proteolytic fragments of JNK1 and JNK3 were observed and protein levels, but not activity, of JNK isoforms increased in a detergent-insoluble fraction. Two novel c-Jun (and ATF-2) kinase activities increased during reperfusion of animals occluded for 60 min. An activity designated p46(slow) was similar in M(r) to a JNK2 isoform induced in these animals. A second 30-kDa activity associated with the detergent-insoluble fraction co-migrated with a JNK3 N-terminal fragment. The results show that JNK1 is active in the normal CNS and increased activity is not associated with durations of ischemia and reperfusion that induce cell death. However, specific JNK isoform activation may participate in the cell death pathways as increased activity of novel c-Jun (ATF-2) kinase activities was observed in paraplegic animals.

A dose response for radiation-induced intrachromosomal DNA rearrangements detected by inverse polymerase chain reaction.

X-ray-induced intrachromosomal DNA rearrangements were detected in the 5' region of the MYC gene of cells of the human bladder carcinoma cell line, EJ-30, by using PCR with inverted primers. When the cells were allowed to repair/misrepair for 6 or 23 h after irradiation, the frequency of rearrangements increased with dose from (0.7 +/- 0.4) x 10(-5) per copy of MYC for unirradiated cells to (3.2 +/- 0.7) x 10(-5) after 30 Gy, (5.4 +/- 1.2) x 10(-5) after 70 Gy, and (5.9 +/- 1.0) x 10(-5) after 100 Gy. No significant difference was observed between 6 and 23 h of repair. Sequences obtained from the products suggest that there was no homology between the two sequences involved in the recombination event and that there was no clustering of breakpoints. The procedure is relatively simple, requiring only one digestion with a rare-cutting restriction enzyme prior to PCR amplification of the DNA purified from irradiated cells. The site of enzyme digestion is located between a pair of primer sites 120 bp apart for which the primers face in opposite directions. If no intrachromosomal rearrangement has occurred, no PCR product would be obtained. However, if an intrachromosomal rearrangement has occurred between two regions located on either side of the primer sites, an episome or duplication event would result if the rearrangement had occurred either within the same chromatid or between two sister chromatids, respectively. Digestion between the primers would linearize an episome or release a linear molecule containing the duplicated primer sites from a larger molecule. After both types of rearrangement events, the primers would be facing each other and would be located on either end of the linear molecule; and if they are less than approximately 5 kb apart, PCR amplification should result in a product. This procedure is relatively simple and rapid and does not require any cell division after irradiation or phenotypic selection of mutants. Also, quantification is based on the number of PCR products detected in a known amount of DNA, and not on a precise determination of the amount of PCR amplification that has occurred. Thus the inverse PCR procedure has the potential ofbeing used as an assay to detect variations in radiation-induced frequencies of DNA rearrangements.

Leptin immunoreactivity in the central nervous system in normal and diabetic rats.

An anti-leptin polyclonal antiserum was used to detect leptin-immunoreactivity (-ir) in the central nervous system in rats. Leptin-ir was found in perikaryon and processes of neurons in the hypothalamus, including the paraventricular nucleus, and in several regions in the brainstem, including the nucleus of the solitary tract. In streptozotocin-treated diabetic rats, leptin-ir was dramatically attenuated only in the hypothalamus, and was restored by insulin treatment. Our findings that removal or restoration of systemic insulin regulates hypothalamic leptin-ir suggests that the leptin-ir observed in the brain is sensitive to peripheral hormone status. The presence of insulin-regulated leptin in the hypothalamus reveals a new avenue of research on the central actions of leptin in regulating energy balance and metabolism.

Simultaneous infection with two strains of Mycobacterium tuberculosis identified by restriction fragment length polymorphism analysis.

Simultaneous infection with two different strains of Mycobacterium tuberculosis has been demonstrated using phage typing. We report here the first case of mixed infection identified using IS6110-based genotyping of M. tuberculosis. The patient was diagnosed with pulmonary tuberculosis in February, 1991. The initial isolate of M. tuberculosis had two different genotype patterns (dark 7-band and light 14-band patterns). However, in a repeat isolate obtained several months later, only the 14-band pattern was visible. Exogenous reinfection and laboratory cross-contamination were unlikely because both genotype patterns were unique in the San Francisco database which includes over 1300 isolates of M. tuberculosis. This case demonstrates the importance of identifying mixed infections in the study of the molecular epidemiology of tuberculosis. Mixed infections could be confused with exogenous reinfection or laboratory cross-contamination, and important epidemiologic connections could be missed.