Dissolved polycyclic aromatic compounds in Canada's Athabasca River in relation to Oil Sands from 2013 through 2019.

The Canada-Alberta Oil Sands Monitoring (OSM) Program began long-term surface water quality monitoring on the lower Athabasca River in 2012. Sampling of low level, bio-accumulative polycyclic aromatic compounds (PACs) targeted a suite of parent and alkylated compounds in the Athabasca River (AR) mainstem using semi-permeable membrane devices (SPMDs). Samples were collected along a gradient from upstream reference near Athabasca, Alberta, through exposure to the Athabasca oil sands deposit (AOSD), various tributary inflows, and mining activities within the OSMA, to downstream recovery near Wood Buffalo National Park (WBNP) and reference on the Slave River. The program adapted over the years, shifting in response to program review and environmental events. The AOSD chemical fingerprint was present in samples collected within the AOSD, through the oil sands mineable area (OSMA), downstream to recovery from 2013 to 2019. PACs were dominated by alkylated phenanthrenes/anthracenes (PAs) and dibenzothiophenes (Ds), with elevated levels of alkylated fluorenes (Fs), naphthalenes (Ns), fluoranthenes/pyrenes (FlPys) and benzo[a]anthracenes/chrysenes (BaACs), increasing in concentration from C1 < C2 < C3 < C4. Concentrations of these petrogenic PACs were at their highest within the OSMA and downstream of tributaries. The AOSD fingerprint was absent from sites located outside of the influence of the AOSD and downstream of the Peace-Athabasca Delta on the Slave River. PAC concentrations in the AR increased with mainstem discharge and loadings from tributaries, were moderated by the PAD, and diluted by the Peace River. This work bolsters the baseline PAC information previously reported for the Athabasca River and waters downstream, reporting 7 years of data, from all sites within the mainstem monitoring program, and exploring potential regional and hydrological drivers of these between sites and over time.

Impact of sample collection on prokaryotic and eukaryotic diversity of niche environments of the oil-sand mining impacted Athabasca River.

Microbial communities are an important aspect of overall riverine ecology; however, appreciation of the effects of anthropogenic activities on unique riverine microbial niches, and how the collection of these samples affects the observed diversity and community profile is lacking. We analyzed prokaryotic and eukaryotic communities from surface water, biofilms, and suspended load niches along a gradient of oil sands-related contamination in the Athabasca River (Alberta, Canada), with suspended load or particle-associated communities collected either via Kenney Sampler or centrifugation manifold. At the phylum level, different niche communities were highly similar to each other and across locations. However, there were significant differences in the abundance of specific genera among the different niches and across sampling locations. A generalized linear model revealed that use of the Kenney Sampler resulted in more diverse bacterial and eukaryotic suspended load community than centrifugal collection, though suspended load communities collected by any means remained stably diverse across locations. Although there was an influence of water quality parameters on community composition, all sampled sites support diverse bacterial and eukaryotic communities regardless of the degree of contamination, highlighting the need to look beyond ecological diversity as a means of assessing ecological perturbations, and consider collecting samples from multiple niche environments.

Costs of Not Getting to Know You: Lower Levels of Parental Reflective Functioning Confer Risk for Maternal Insensitivity and Insecure Infant Attachment.

Parental reflective functioning (PRF) is a robust predictor of parenting sensitivity and secure infant attachment, but its assessment requires extensive resources, limiting its integration into research and clinical practice. The Mini-Parent Reflective Functioning Interview (Mini-PRFI) assesses the parent's capacity to mentalize for his/her 6-month-old infant (rated using the PRF coding system; Slade et al., 2004, PRF coding system and Slade REF, Unpublished protocol, New York, NY: The City University of New York). In the current study, we examined whether Mini-PRFI scores were associated with theoretically related constructs; to establish a point of comparison, we evaluated links between Mini-PRFI scores alongside RF assessed from the Adult Attachment Interview (AAI). Mother-infant dyads (N = 88) completed the AAI before the birth of the infant, the Mini-PRFI and an interaction task (rated for insensitive parental behavior) when infants were 6 months old, as well as the Strange Situation Procedure when infants were 16 months old. Mini-PRFI scores were strongly positively associated with AAI RF and negatively associated with maternal insensitivity. Mini-PRFI scores predicted infant attachment organization (secure/insecure, organized/disorganized) at 16 months, and this effect was mediated by parenting insensitivity. These findings suggest that the Mini-PRFI predicts theoretically related attachment constructs, demonstrating the promise of the Mini-PRFI to increase the accessibility of interview-based PRF measurements to clinicians and researchers.

Modularization and epistatic hierarchy determine homeostatic actions of multiple blood pressure quantitative trait loci.

Hypertension, the most frequently diagnosed clinical condition world-wide, predisposes individuals to morbidity and mortality, yet its underlying pathological etiologies are poorly understood. So far, a large number of quantitative trait loci (QTLs) have been identified in both humans and animal models, but how they function together in determining overall blood pressure (BP) in physiological settings is unknown. Here, we systematically and comprehensively performed pair-wise comparisons of individual QTLs to create a global picture of their functionality in an inbred rat model. Rather than each of numerous QTLs contributing to infinitesimal BP increments, a modularized pattern arises: two epistatic 'blocks' constitute basic functional 'units' for nearly all QTLs, designated as epistatic module 1 (EM1) and EM2. This modularization dictates the magnitude and scope of BP effects. Any EM1 member can contribute to BP additively to that of EM2, but not to those of the same module. Members of each EM display epistatic hierarchy, which seems to reflect a related functional pathway. Rat homologues of 11 human BP QTLs belong to either EM1 or EM2. Unique insights emerge into the novel genetic mechanism and hierarchy determining BP in the Dahl salt-sensitive SS/Jr (DSS) rat model that implicate a portion of human QTLs. Elucidating the pathways underlying EM1 and EM2 may reveal the genetic regulation of BP.

Aerobic biofilms grown from Athabasca watershed sediments are inhibited by increasing concentrations of bituminous compounds.

Sediments from the Athabasca River and its tributaries naturally contain bitumen at various concentrations, but the impacts of this variation on the ecology of the river are unknown. Here, we used controlled rotating biofilm reactors in which we recirculated diluted sediments containing various concentrations of bituminous compounds taken from the Athabasca River and three tributaries. Biofilms exposed to sediments having low and high concentrations of bituminous compounds were compared. The latter were 29% thinner, had a different extracellular polysaccharide composition, 67% less bacterial biomass per µm2, 68% less cyanobacterial biomass per µm2, 64% less algal biomass per µm2, 13% fewer protozoa per cm2, were 21% less productive, and had a 33% reduced content in chlorophyll a per mm2 and a 20% reduction in the expression of photosynthetic genes, but they had a 23% increase in the expression of aromatic hydrocarbon degradation genes. Within the Bacteria, differences in community composition were also observed, with relatively more Alphaproteobacteria and Betaproteobacteria and less Cyanobacteria, Bacteroidetes, and Firmicutes in biofilms exposed to high concentrations of bituminous compounds. Altogether, our results suggest that biofilms that develop in the presence of higher concentrations of bituminous compounds are less productive and have lower biomass, linked to a decrease in the activities and abundance of photosynthetic organisms likely due to inhibitory effects. However, within this general inhibition, some specific microbial taxa and functional genes are stimulated because they are less sensitive to the inhibitory effects of bituminous compounds or can degrade and utilize some bitumen-associated compounds.

Prevalence and risk of potential cytochrome P450-mediated drug-drug interactions in older hospitalized patients with polypharmacy.

BACKGROUND: As rates of polypharmacy rise and medication regimens become more complex, the risk of potential cytochrome P450 (CYP)-mediated drug-drug interactions (DDIs) is a growing clinical concern for older adults. OBJECTIVE: To determine the prevalence of potential CYP-mediated DDIs in older hospitalized adults with polypharmacy and analyze the relationship between the number of drugs dispensed and the probability of these interactions in this high-risk population. METHODS: A prospective 16-week cohort study was conducted among consecutive new patients aged 65 years and older with polypharmacy (>5 drugs) admitted to a community hospital. The medication profiles of these patients were analyzed with a new multidrug cytochrome-specific software program. The prevalence of potential CYP-mediated DDIs was determined, with the probability calculated as a function of the number of medications dispensed using multivariate Poisson regression adjusted for age and sex. Comparative performance of the software program and a standard 2-drug alert program for detecting these DDIs was evaluated using the Wilcoxon-Mann-Whitney rank-sum test. Pharmacists' decisions to recommend medication adjustment based on the probability of CYP-mediated DDIs were recorded. RESULTS: The prevalence of potential CYP-mediated DDIs detected among 275 older adults with polypharmacy was 80%. The probability of at least 1 CYP-mediated DDI was 50% for persons taking 5-9 drugs, 81% with 10-14 drugs, 92% with 15-19 drugs, and 100% with 20 or more drugs. Addition of each medication to a 5-drug regimen conferred a 12% increased risk of a potential CYP-mediated DDI after adjustment for age and sex (OR 1.12; 95% CI 1.09-1.14). The multidrug software identified a median increase of 3 (95% CI 2.5-3.5) potential CYP-mediated DDIs per patient, compared to use of the standard 2-drug alert software. Pharmacists targeted patients for medication adjustment or close clinical monitoring in 23% of cases. CONCLUSIONS: The prevalence of potential CYP-mediated DDIs is high in geriatric patients with polypharmacy. The risk of DDIs increases as a function of the number of medications dispensed. Pharmacists' decision to intervene for potential CYP-mediated DDIs depends on clinical judgment in addition to the output from drug alert software programs, but may be facilitated by a single multicomponent, multidrug potential CYP-mediated DDI assessment.

Artificial neural network modeling for drug dialyzability prediction.

PURPOSE: The purpose of this study was to develop an artificial neural network (ANN) model to predict drug removal during dialysis based on drug properties and dialysis conditions. Nine antihypertensive drugs were chosen as model for this study. METHODS: Drugs were dissolved in a physiologic buffer and dialysed in vitro in different dialysis conditions (UFRmin/UFRmax, with/without BSA). Samples were taken at regular intervals and frozen at -20ºC until analysis. Extraction methods were developed for drugs that were dialysed with BSA in the buffer. Drug concentrations were quantified by high performance liquid chromatography (HPLC) or mass spectrometry (LC/MS/MS). Dialysis clearances (CLDs) were calculated using the obtained drug concentrations. An ANOVA with Scheffe's pairwise adjustments was performed on the collected data in order to investigate the impact of drug plasma protein binding and ultrafiltration rate (UFR) on CLD. The software Neurosolutions was used to build ANNs that would be able to predict drug CLD (output). The inputs consisted of dialysis UFR and the herein drug properties: molecular weight (MW), logD and plasma protein binding. RESULTS: Observed CLDs were very high for the majority of the drugs studied. The addition of BSA in the physiologic buffer statistically significantly decreased CLD for carvedilol (p= 0.002) and labetalol (p<0.001), but made no significant difference for atenolol (p= 0.100). In contrast, varying UFR does not significantly affect CLD (p>0.025). Multiple ANNs were built and compared, the best model was a Jordan and Elman network which showed learning stability and good predictive results (MSEtesting = 129). CONCLUSION: In this study, we have developed an ANN-model which is able to predict drug removal during dialysis. Since experimental determination of all existing drug CLDs is not realistic, ANNs represent a promising tool for the prediction of drug CLD using drug properties and dialysis conditions.

Prediction of in vivo atenolol removal by high-permeability hemodialysis based on an in vitro model.

PURPOSE: In order to update our data on drug dialyzability using the high-permeability dialysis membranes, atenolol elimination by an in vitro dialysis model was compared to that observed in six patients during high-permeability hemodialysis (HD), and the predictive value of the model was evaluated. METHODS: Atenolol clearance was evaluated in six patients undergoing chronic HD. They were considered as eligible candidates if they were between 18 and 80 years of age, had a body mass index between 19 and 30 kg/m2, underwent HD and were taking atenolol on a regular basis in oral tablet form for at least 1 month before the study started. Atenolol clearance was also evaluated in three in vitro dialysis sessions with high-permeability polysulfone membrane. Atenolol was dissolved in 6 L of Krebs-Henseleit buffer with bovine serum albumin. Dialysis parameters were set to mirror as much as possible the patients' parameters (flow rate: 300 mL/min, dialyzate flow: 500 mL/min). After sample collection, drug concentrations were measured with high performance liquid chromatography. The comparison between in vivo and in vitro atenolol elimination kinetics was performed by drawing the curve fittings of concentrations vs. time on SigmaPlot 12, and adding a 95% prediction interval to each elimination curve fitting. RESULTS: Mean dialysis clearance of atenolol in vitro and in vivo was 198 ± 4 and 235 ± 53 mL/min, respectively. Atenolol was significantly removed within the study time period in both in vitro and in vivo experiments. By the end of in vitro dialysis, atenolol remaining in the drug reservoir was less than 2% of initial arterial concentration. CONCLUSION: Our study has indicated that atenolol is almost entirely cleared during high-permeability hemodialysis. Furthermore, the in vitro prediction interval of the drug elimination curve fitting could forecast its in vivo elimination especially at the end of dialysis.

Reactions of atomic metal anions in the gas phase: competition between electron transfer, proton abstraction and bond activation.

Bare metal anions K(-), Rb(-), Cs(-), Fe(-), Co(-), Ni(-), Cu(-), and Ag(-), generated by electrospray ionization of the corresponding oxalate or tricarballylate solutions, were allowed to react with methyl and ethyl chloride, methyl bromide, nitromethane, and acetonitrile in the collision hexapole of a triple-quadrupole mass spectrometer. Observed reactions include (a) the formation of halide, nitride, and cyanide anions, which was shown to be likely due to the insertion of the metal into the C-X, C-N, and C-C bonds, (b) transfer of H(+) from the organic molecule, which is demonstrated to most likely be due to the simple transfer of a proton to form neutral metal hydride, and (c) in the case of nitromethane, direct electron transfer to form the nitromethane radical anion. Interestingly, Co(-) was the only metal anion to transfer an electron to acetonitrile. Differences in the reactions are related to the differences in electron affinity of the metals and the Δ(acid)H° of the metals and organic substrates. Density functional theory calculations at the B3-LYP/6-311++G(3df,2p)//B3-LYP/6-31+G(d) level of theory shed light on the relative energetics of these processes and the mechanisms by which they take place.

Investigation on gene transfer from genetically modified corn (Zea mays L.) plants to soil bacteria.

Knowledge about the prevalence and diversity of antibiotic resistance genes in soil bacteria communities is required to evaluate the possibility and ecological consequences of the transfer of these genes carried by genetically modified (GM) plants to soil bacteria. The neomycin phosphotransferase gene (nptII) conferring resistance to kanamycin and neomycin is one of the antibiotic resistance genes commonly present in GM plants. In this study, we investigated kanamycin-resistant (Km(R)) and neomycin-resistant (Nm(R)) soil bacterial populations in a 3-year field trial using a commercial GM corn (Zea mays L.) carrying the nptII gene and its near isogenic line. The results showed that a portion (2.3 - 15.6 %) of cultivable soil bacteria was naturally resistant to kanamycin or neomycin. However, no significant difference in the population level of Km(R) or Nm(R) soil bacteria was observed between the GM and non-GM corn fields. The nptII gene was not detected in any of the total 3000 Km(R) or Nm(R) isolates screened by PCR. Further, total soil bacterial cells were collected through Nycodenz gradient centrifugation and bacterial community DNA was subjected to PCR. Detection limit was about 500 cells per gram of fresh soil. Our study suggests that the nptII gene was relatively rare in the soil bacterial populations and there was no evidence of gene transfer from a GM corn plant to soil bacteria based on the data from total soil bacterial communities.

Long-timescale molecular-dynamics simulations of the major urinary protein provide atomistic interpretations of the unusual thermodynamics of ligand binding.

The mouse major urinary protein (MUP) has proved to be an intriguing test bed for detailed studies on protein-ligand recognition. NMR, calorimetric, and modeling investigations have revealed that the thermodynamics of ligand binding involve a complex interplay between competing enthalpic and entropic terms. We performed six independent, 1.2 micros molecular-dynamics simulations on MUP--three replicates on the apo-protein, and three on the complex with the pheromone isobutylmethoxypyrazine. Our findings provide the most comprehensive picture to date of the structure and dynamics of MUP, and how they are modulated by ligand binding. The mechanical pathways by which amino acid side chains can transmit information regarding ligand binding to surface loops and either increase or decrease their flexibility (entropy-entropy compensation) are identified. Dewetting of the highly hydrophobic binding cavity is confirmed, and the results reveal an aspect of ligand binding that was not observed in earlier, shorter simulations: bound ligand retains extensive rotational freedom. Both of these features have significant implications for interpretations of the entropic component of binding. More generally, these simulations test the ability of current molecular simulation methods to produce a reliable and reproducible picture of protein dynamics on the microsecond timescale.

Acrolein induces apoptosis through the death receptor pathway in A549 lung cells: role of p53.

Acrolein, a highly reactive alpha,beta-unsaturated aldehyde, is an omnipresent environmental pollutant. Chronic and acute human exposures occur through exogenous and endogenous sources, including food, vapors of overheated cooking oil, house and forest fires, cigarette smoke, and automobile exhaust. Acrolein is a toxic byproduct of lipid peroxidation, which has been implicated in pulmonary, cardiac, and neurodegenerative diseases. This study shows that p53 is an initiating factor in acrolein-induced death receptor activation during apoptosis in A549 human lung cells. Exposure of cells to acrolein (0-50 micromol/L) mainly caused apoptosis, which was manifested by execution phase events such as condensation of nuclear chromatin, phosphatidylserine externalization, and poly(ADP-ribose) polymerase (PARP) cleavage. Levels of necrosis (approximately 5%) were low. Acrolein triggered the death receptor pathway of apoptosis, causing elevation of Fas ligand (FasL) and translocation of adaptor protein Fas-associated death domain to the plasma membrane. Acrolein caused activation of caspase-8, caspase-2, caspase-7, and the cross-talk pathway mediated by Bid cleavage. Activation of p53 and increased expression of p53-upregulated modulator of apoptosis (PUMA) occurred in response to acrolein. FasL upregulation and caspase-8 activation were decreased by p53 inhibitor pifithrin-alpha and antioxidant polyethylene glycol catalase. These findings increase our knowledge about the induction of cell death pathways by acrolein, which has important implications for human health.

Metatranscriptomic Insights Into the Response of River Biofilm Communities to Ionic and Nano-Zinc Oxide Exposures.

Manufactured Zn oxide nanoparticle (ZnO-NP) are extensively used world-wide in personal care and industrial products and are important contaminants of aquatic environments. To understand the overall impact of ZnO-NP contamination on aquatic ecosystems, investigation of their toxicity on aquatic biofilms is of particular consequence, given biofilms are known sinks for NP contaminants. In order to assess alterations in the functional activity of river microbial biofilm communities as a result of environmentally-relevant ZnO-NP exposure, biofilms were exposed to ionic zinc salt or ZnOPs that were uncoated (hydrophilic), coated with silane (hydrophobic) or stearic acid (lipophilic), at a total concentration of 188 µg l-1 Zn. ICP-MS analyses of biofilms indicated ZnO-NP concentrated in the biofilms, with hydrophilic, hydrophobic, and lipophilic treatments reaching 0.310, 0.250, and 0.220 µg Zn cm-2 of biofilm, respectively, while scanning transmission X-ray microspectroscopy (STXM) analyses of biofilms confirmed that Zn was extensively- and differentially-sorbed to biofilm material. Microbial community composition, based on taxonomic affiliation of mRNA sequences and enumeration of protozoa and micrometazoa, was not affected by these treatments, and the total transcriptional response of biofilms to all experimental exposures was not indicative of a global toxic-response, as cellular processes involved in general cell maintenance and housekeeping were abundantly transcribed. Transcripts related to major biological processes, including photosynthesis, energy metabolism, nitrogen metabolism, lipid metabolism, membrane transport, antibiotic resistance and xenobiotic degradation, were differentially expressed in Zn-exposures relative to controls. Notably, transcripts involved in nitrogen fixation and photosynthesis were decreased in abundance in response to Zn-exposure, while transcripts related to lipid degradation and motility-chemotaxis were increased, suggesting a potential role of Zn in biofilm dissolution. ZnO-NP and ionic Zn exposures elicited generally overlapping transcriptional responses, however hydrophilic and hydrophobic ZnO-NPs induced a more distinct effect than that of lipophilic ZnO-NPs, which had an effect similar to that of low ionic Zn exposure. While the physical coating of ZnO-NP may not induce specific toxicity observable at a community level, alteration of ecologically important processes of photosynthesis and nitrogen cycling are an important potential consequence of exposure to ionic Zn and Zn oxides.

Microscale and molecular analyses of river biofilm communities treated with microgram levels of cerium oxide nanoparticles indicate limited but significant effects.

Cerium oxide (CeO2) nanoparticles are used as in-fuel catalysts and in manufacturing processes, creating a potential for release to aquatic environments. Exposures at 1 and 10 µg/L CeO2-nanoparticles were made to assess effects during the development of river biofilm communities. Scanning transmission x-ray microscopy (STXM) indicated extensive sorption of nanoparticles to the community and co-localization with lipid moieties. Following 8 weeks of development, polycarbonate coupons were removed from the reactors and used for molecular analyses, denaturing gradient gel electrophoresis analysis (DGGE-16S rRNA) and 16S rRNA amplicon sequencing. Microscopic imaging of the biofilm communities (bacterial, photosynthetic biomass, exopolymer composition, thickness, protozoan numbers), as well as carbon substrate utilization fingerprinting was performed. There was a trend toward reduced photosynthetic biomass, but no significant effects of CeO2 exposure were found on photosynthetic and bacterial biomass or biofilm thickness. Sole carbon source utilization analyses indicated increased utilization of 10 carbon sources in the carbohydrate, carboxylic acid and amino acids categories related to CeO2 exposures; however, predominantly, no significant effects (p < 0.05) were detected. Measures of microbial diversity, lectin binding affinities of exopolymeric substances and results of DGGE analyses, indicated significant changes to community composition (p < 0.05) with CeO2 exposure. Increased binding of the lectin Canavalia ensiformis was observed, consistent with changes in bacterial-associated polymers. Whereas, no significant changes were observed in binding to residues associated with algal and cyanobacterial exopolymers. 16S rRNA amplicon sequencing of community DNA indicated changes in diversity and shifts in community composition; however, these did not trend with increasing CeO2 exposure. Counting of protozoans in the biofilm communities indicated no significant effects on this trophic level. Thus, based on biomass and functional measures, CeO2 nanoparticles did not appear to have significant effects; however, there was evidence of selection pressure resulting in significant changes in microbial community composition.

N,N-Diethyl-m-Toluamide Exposure at an Environmentally Relevant Concentration Influences River Microbial Community Development.

Studies of the South Saskatchewan River confirmed that N,N-diethyl-m-toluamide (DEET) is ubiquitous at 10 to 20 ng/L, whereas in effluent-dominated Wascana Creek, levels of 100 to 450 ng/L were observed. Effects of DEET exposure were assessed in microbial communities using a wide variety of measures. Communities developed in rotating annular reactors with either 100 or 500 ng/L DEET, verified using gas chromatography-mass spectrometry analyses. Microscale analyses indicated that both DEET concentrations resulted in significant (p < 0.05) declines in photosynthetic biomass, whereas bacterial biomass was unaffected. There was no detectable effect of DEET on the levels of chlorophyll a. However, pigment analyses indicated substantial shifts in algal-cyanobacterial community structure, with reductions of green algae and some cyanobacterial groups at 500 ng/L DEET. Protozoan/micrometazoan grazers increased in communities exposed to 500 ng/L, but not 100 ng/L, DEET. Based on thymidine incorporation or utilization of carbon sources, DEET had no significant effects on metabolic activities. Fluorescent lectin-binding analyses showed significant (p < 0.05) changes in glycoconjugate composition at both DEET concentrations, consistent with altered community structure. Principal component cluster analyses of denaturing gradient gel electrophoresis indicated that DEET exposure at either concentration significantly changed the bacterial community (p < 0.05). Analyses based on 16S ribosomal RNA of community composition confirmed changes with DEET exposure, increasing detectable beta-proteobacteria, whereas actinobacteria and acidimicrobia became undetectable. Further, cyanobacteria in the subclass Oscillatoriophycideae were similarly not detected. Thus, DEET can alter microbial community structure and function, supporting the need for further evaluation of its effects in aquatic habitats. Environ Toxicol Chem 2019;38:2414-2425. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

A nuclease-resistant RNA aptamer specifically inhibits angiopoietin-1-mediated Tie2 activation and function.

Tie2 is a receptor tyrosine kinase that is expressed predominantly in the endothelium and plays key roles in both physiological and pathological angiogenesis. The ligands for Tie2, the angiopoietins (Ang), perform opposing functions in vascular maintenance and angiogenesis; Ang1 regulates vascular quiescence, while Ang2 is thought to promote vascular destabilization and facilitate angiogenesis. However, the mechanisms responsible for these differences are not understood. To begin to elucidate the molecular differences between the angiopoietins, we previously developed a specific RNA aptamer inhibitor of Ang2. Here, we used the same iterative in vitro selection process, termed SELEX (Systematic Evolution of Ligands by EXponential enrichment), to screen a library of 2'-fluoro-modified ribonucleotides for Ang1-binding aptamers. After nine rounds of selection, we identified a single clone, ANG9-4, that bound with high affinity to human Ang1 (K ( d ) 2.8 nM) but not Ang2 (K ( d ) > 1 microM), demonstrating specificity for Ang1. ANG9-4 blocked Ang1-mediated Tie2 phosphorylation and downstream Akt activation. Moreover, ANG9-4 inhibited Ang1-induced endothelial cell survival. Together, these findings demonstrate the feasibility of developing an Ang1-inhibitory aptamer. ANG9-4 and its derivatives may provide useful tools for elucidating the biology of Ang1 and for treating certain angiogenic diseases.

Submegabase resolution of epistatically interacting quantitative trait loci for blood pressure applicable for essential hypertension.

OBJECTIVE: Although genetic mapping of quantitative trait loci for blood pressure to large chromosome segments is readily achievable, their final identification confronts formidable hurdles. Restriction of the genes lodging in one quantitative trait locus interval to experimental limitation can facilitate their positional cloning. We previously delineated several quantitative trait loci for blood pressure on chromosome 10 of Dahl salt-sensitive rats, but their chromosome delimitations were either large or not definitive. METHODS: In this study, we systematically and comprehensively constructed congenic strains with submegabase (Mb) genome resolution and analyzed their blood pressure by telemetry. RESULTS: Three quantitative trait loci have been conclusively delimited by three congenic strains, each independently lowering the blood pressure. Their intervals are demarcated by genomic regions between 350 and 910 kilobases (kb) in size. Two of the three quantitative trait loci share an epistatic relationship and are separated from one another by less than 170 kb. Two additional quantitative trait loci for blood pressure were also tentatively delineated and their intervals range from 520 kb to 1.75 Mb. Possible genes dwelling in each quantitative trait locus-interval number between 11 and 17. None of these genes is known to exert a functional impact on blood pressure. Work is underway to find candidate genes with mutations that could be responsible for the blood pressure effect. CONCLUSION: Novel diagnostic, prognostic, preventive and/or therapeutic targets for essential hypertension and hypertension-associated diseases are likely to emerge from the identification of these quantitative trait loci. Potential applications of these quantitative trait loci to humans are suggested from the positive results from several association studies, demonstrating the existence of quantitative trait loci in the broad homologous regions.

Distinct genomic replacements from Lewis correct diastolic dysfunction, attenuate hypertension, and reduce left ventricular hypertrophy in Dahl salt-sensitive rats.

BACKGROUND: Hypertension and diastolic heart failure are two common cardiovascular diseases that inflict heavy morbidity and mortality, yet relatively little is understood about their pathophysiology. The identification of quantitative trait loci for blood pressure is important in unveiling the causes of polygenic hypertension. Although Dahl salt-sensitive strain is also an excellent model for the study of diastolic heart failure, virtually nothing is known about the quantitative trait loci determining diastolic heart failure. Diastolic dysfunction often represents the onset of diastolic heart failure. METHODS: We first characterized the cardiac phenotype of Dahl salt-sensitive strain and normotensive Lewis control rats by echocardiography to ascertain diastolic function. We then analyzed corresponding features of four newly developed and two existing congenic strains, each of which carries a specific chromosome substitution of Dahl salt-sensitive strain by its Lewis homologue and each lowering blood pressure. RESULTS: Dahl salt-sensitive strain displayed diastolic dysfunction that was rectified in two of six congenic strains, designated as positive congenic strains, which represent the first rodent models exhibiting functional normalization of diastolic dysfunction caused by naturally occurring genetic variants. The two positive congenic strains also showed a reduction in left ventricular mass. In contrast, four of six congenic strains did not change diastolic function despite their blood pressure-lowering effects. CONCLUSION: Genes present in the replaced chromosome segments of the two positive congenic strains are not commonly known to affect blood pressure, diastolic function or left ventricular mass. Consequently, novel prognostic, diagnostic and therapeutic strategies for hypertensive diastolic heart failure likely emerge from this work.

Mechanism of cell death induced by spermine and amine oxidase in mouse melanoma cells.

Polyamines such as spermine, spermidine and putrescine are necessary for cell proliferation and are detected at higher concentrations in most tumor tissues, compared to normal tissues. The amine oxidase enzymes can generate cytotoxic products such as hydrogen peroxide and aldehydes from these polyamines. This study investigates the mechanisms of cell death in B16-F0 mouse melanoma tumor cells exposed to bovine serum amine oxidase and exogenous spermine. The bovine serum amine oxidase/spermine enzymatic system induced inhibition of cell proliferation in B16-F0 melanoma cells and cell death by both apoptotic and necrotic processes. Bovine serum amine oxidase or spermine, alone, did not induce cytotoxicity or cell death by apoptosis, indicating that the enzymatic reaction products were responsible. Catalase and NAD-dependent aldehyde dehydrogenase, inhibitors of hydrogen peroxide and aldehydes, respectively, decreased cell death by apoptosis and necrosis. This further confirms that the cytotoxic products are responsible for causing cell death. Use of inhibitors of different caspases showed that melanoma cells were sensitive to processes involving caspase-3 and -9, but were insensitive to caspase-6. Bovine serum amine oxidase in the presence of spermine could be useful as a promising new tool for anticancer treatment by the selective generation of toxic compounds from polyamines in tumors.