Publications

OBJECTIVE

The purpose of this series was to explore a 12.5:1 fixed-dose ratio of an intravenous nalbuphine and naloxone mixture (NNM) for use in patients following gynecologic surgery.

DESIGN AND PATIENTS

Open-label, nonrandomized case series. The first series was a dose-ranging investigation for 12 patients following elective total abdominal hysterectomy or myomectomy. In this series, fentanyl was used for intraoperative analgesia, and patients were assigned to a lower NNM (2.5 mg/0.2 mg) or to a higher NNM (5 mg/0.4 mg) dose group. The second series evaluated the fixed dose of 5 mg nalbuphine/0.4 mg naloxone for four patients undergoing ambulatory gynecologic procedures. In the second series, no opioid agents were administered intraoperatively to eliminate the possibility of mu-opioid reversal by naloxone postoperatively.

OUTCOME MEASURES

Pain control was assessed using a Verbal Pain Scale (0-10). Vital signs, side effects, and adverse events were recorded to determine drug safety.

RESULTS

In the first series, there were no adverse events; however, each patient required rescue medication (either morphine or fentanyl). In the second series, two of the four patients reported a reduction in pain following drug administration and did not require any further analgesic agents in the 3-hour postoperative period. One patient had an asymptomatic lowering of heart rate after receiving the drug.

CONCLUSION

Additional research of the unique combination therapy of nalbuphine and naloxone is warranted to further determine its potential clinical efficacy and safety.

Existing methods to ascertain small sets of markers for the identification of human population structure require prior knowledge of individual ancestry. Based on Principal Components Analysis (PCA), and recent results in theoretical computer science, we present a novel algorithm that, applied on genomewide data, selects small subsets of SNPs (PCA-correlated SNPs) to reproduce the structure found by PCA on the complete dataset, without use of ancestry information. Evaluating our method on a previously described dataset (10,805 SNPs, 11 populations), we demonstrate that a very small set of PCA-correlated SNPs can be effectively employed to assign individuals to particular continents or populations, using a simple clustering algorithm. We validate our methods on the HapMap populations and achieve perfect intercontinental differentiation with 14 PCA-correlated SNPs. The Chinese and Japanese populations can be easily differentiated using less than 100 PCA-correlated SNPs ascertained after evaluating 1.7 million SNPs from HapMap. We show that, in general, structure informative SNPs are not portable across geographic regions. However, we manage to identify a general set of 50 PCA-correlated SNPs that effectively assigns individuals to one of nine different populations. Compared to analysis with the measure of informativeness, our methods, although unsupervised, achieved similar results. We proceed to demonstrate that our algorithm can be effectively used for the analysis of admixed populations without having to trace the origin of individuals. Analyzing a Puerto Rican dataset (192 individuals, 7,257 SNPs), we show that PCA-correlated SNPs can be used to successfully predict structure and ancestry proportions. We subsequently validate these SNPs for structure identification in an independent Puerto Rican dataset. The algorithm that we introduce runs in seconds and can be easily applied on large genome-wide datasets, facilitating the identification of population substructure, stratification assessment in multi-stage whole-genome association studies, and the study of demographic history in human populations.

BACKGROUND

OCTN1 is a multispecific transporter of organic cations and zwitterions, including several clinically important drugs as well as the antioxidant ergothioneine. OCTN1 is highly expressed in the kidney, where it is thought to aid in active secretion of organic cations, and may facilitate the active reabsorption of ergothioneine. Genetic variation in OCTN1 may help to explain interindividual variability in the pharmacokinetics of many cationic or zwitterionic drugs.

METHODS

We screened for human genetic variants in the OCTN1 coding region by direct sequencing in a large sample (n=270) of ethnically diverse healthy volunteers.

RESULTS

Six protein sequence-altering variants were identified, including five-amino-acid substitutions and one nonsense mutation. Two of the variants, T306I and L503F, were polymorphic, occurring at frequencies of 37 and 19%, respectively, in the total sample. Allele frequencies are varied by ethnicity. In biochemical assays, two of the variants (D165G and R282X) resulted in complete loss of transport function, and one variant (M205I) caused a reduction in activity to approximately 50% of the reference sequence protein. One variant, L503F, showed altered substrate specificity; this variant occurred at particularly high allele frequency (42%) in the European-American participants in our sample. Subcellular localization and ergothioneine inhibition kinetics were similar among the common amino-acid sequence variants of OCTN1.

CONCLUSIONS

The common OCTN1-L503F variant may explain a significant amount of population variation in the pharmacokinetics of OCTN1 substrate drugs. The rare loss-of-function variants provide a rational tool for studying the importance of ergothioneine in humans in vivo.

Ultraconserved elements have been suggested to retain extended perfect sequence identity between the human, mouse, and rat genomes due to essential functional properties. To investigate the necessities of these elements in vivo, we removed four noncoding ultraconserved elements (ranging in length from 222 to 731 base pairs) from the mouse genome. To maximize the likelihood of observing a phenotype, we chose to delete elements that function as enhancers in a mouse transgenic assay and that are near genes that exhibit marked phenotypes both when completely inactivated in the mouse and when their expression is altered due to other genomic modifications. Remarkably, all four resulting lines of mice lacking these ultraconserved elements were viable and fertile, and failed to reveal any critical abnormalities when assayed for a variety of phenotypes including growth, longevity, pathology, and metabolism. In addition, more targeted screens, informed by the abnormalities observed in mice in which genes in proximity to the investigated elements had been altered, also failed to reveal notable abnormalities. These results, while not inclusive of all the possible phenotypic impact of the deleted sequences, indicate that extreme sequence constraint does not necessarily reflect crucial functions required for viability.

OBJECTIVE

Colorectal cancer (CRC) screening uptake remains poor. Until we understand patient motivation and preferences for undertaking screening, it is unlikely the uptake will be optimal. Our objective is to examine patient preferences for CRC screening modalities and uptake rates using utility-based methods.

METHODS

The preference survey was mailed to a random sample of Canadian subjects aged 40 to 60 years from a primary care network. A fractional factorial experimental design maximized D-efficiency and included four blocks with 12 choice tasks in a conditional two-step design, two-alternative discrete choice format with five screening attributes (process, pain, preparation, sensitivity, and specificity). Bivariate probit regression analysis was used to estimate patient preferences for attributes, choice probabilities for alternative modalities and expected rates of uptake.

RESULTS

Five hundred forty-seven of 1047 surveys were returned. Almost 30% of respondents preferred no screening. The most preferred test attribute levels were noninvasive process (e.g., CT), no preparation, no pain, 100% specificity, and 90% sensitivity. Accuracy-related attributes were more important than test process-related attributes. Virtual colonoscopy was the most preferred, followed by colonoscopy, barium enema, sigmoidoscopy, and fecal DNA testing, based on simulated choice probability estimates. Fecal occult blood testing (FOBT) was least preferred. Adjusted screening uptake rate estimates showed the greatest impact (42% increase) would be achieved if all CRC screening modalities were available rather than FOBT alone.

CONCLUSIONS

Our findings emphasize the important role of patient preferences for no screening and in selecting alternative CRC screening modalities. CRC screening implementation in Canada should consider patient preferences to optimize uptake.

A very potent and specific inhibitor of mitochondrial NADH:ubiquinone oxidoreductase (complex I), a derivative of NADH (NADH-OH) has recently been discovered (Kotlyar, A. B., Karliner, J. S., and Cecchini, G. (2005) FEBS Lett. 579, 4861-4866). Here we present a quantitative analysis of the interaction of NADH-OH and other nucleotides with oxidized and reduced complex I in tightly coupled submitochondrial particles. Both the rate of the NADH-OH binding and its affinity to complex I are strongly decreased in the presence of succinate. The effect of succinate is completely reversed by rotenone, antimycin A, and uncoupler. The relative affinity of ADP-ribose, a competitive inhibitor of NADH oxidation, is also shown to be significantly affected by enzyme reduction (KD of 30 and 500 microM for oxidized and the succinate-reduced enzyme, respectively). Binding of NADH-OH is shown to abolish the succinate-supported superoxide generation by complex I. Gradual inhibition of the rotenone-sensitive uncoupled NADH oxidase and the reverse electron transfer activities by NADH-OH yield the same final titration point (approximately 0.1 nmol/mg of protein). The titration of NADH oxidase appears as a straight line, whereas the titration of the reverse reaction appears as a convex curve. Possible models to explain the different titration patterns for the forward and reverse reactions are briefly discussed.

Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that serves as a key regulator of cellular differentiation and survival. Immune stimuli increase S1P synthesis and secretion by mast cells and platelets, implicating this molecule in tissue responses to injury and inflammation. Binding of S1P to G(i) protein-coupled receptors activates phosphatidylinositol 3-kinase and Akt in a variety of tissues. To elucidate the mechanisms by which S1P enhances adult cardiac myocyte survival during hypoxia, we used a mouse cell culture system in which S1P(1) receptors were observed to transduce signals from exogenous S1P, an S1P(1) receptor antibody with agonist properties, and the pharmacological agents FTY720 and SEW2871. S1P(1) receptor mRNA and protein were abundantly expressed by adult mouse cardiac myocytes. S1P-S1P(1) receptor axis enhancement of myocyte survival during hypoxia was abolished by phosphatidylinositol 3-kinase inhibition. S1P(1) receptor function was closely associated with activation of Akt, inactivation of GSK-3beta, and reduction of cytochrome c release from heart mitochondria. These observations highlight the importance of S1P(1) receptors on ventricular myocytes as mediators of inducible resistance against cellular injury during severe hypoxic stress.

The field of tissue engineering has yielded several successes in early clinical trials of regenerative medicine using living cells seeded into biodegradable scaffolds. In contrast to methods that combine biomaterials with living cells, we have developed an approach that uses culture surfaces grafted with the temperature-responsive polymer poly(N-isoproplyacrylamide) that allows for controlled attachment and detachment of living cells via simple temperature changes. Using cultured cell sheets harvested from temperature-responsive surfaces, we have established cell sheet engineering to create functional tissue sheets to treat a wide range of diseases from corneal dysfunction to esophageal cancer, tracheal resection, and cardiac failure. Additionally, by exploiting the unique ability of cell sheets to generate three-dimensional tissues composed of only cultured cells and their deposited extracellular matrix, we have also developed methods to create thick vascularized tissues as well as, organ-like systems for the heart and liver. Cell sheet engineering therefore provides a novel alternative for regenerative medicine approaches that require the re-creation of functional tissue structures.