Monounsaturated fatty acid, carbohydrate intake, and diabetes status are associated with arterial pulse pressure

Background Diabetes is a global epidemic. Cardiovascular disease (CVD) is one of the most prevalent consequences of diabetes. Nutrition is considered a modifiable risk factor for CVD, particularly for individuals with diabetes; albeit, there is little consensus on the role of carbohydrates, proteins and fats for arterial health for persons with or without diabetes. In this study, we examined the association of macronutrients with arterial pulse pressure (APP), a surrogate measure of arterial health by diabetes status and race. Methods Participants were 892 Mexican Americans (MA), 1059 Black, non-Hispanics (BNH) and 2473 White, non-Hispanics (WNH) with and without diabetes of a weighted sample from the National Nutrition and Health Examination Survey (NHANES) 2007-2008. The cross-sectional analysis was performed with IBM-SPSS version 18 with the complex sample analysis module. The two-year sample weight for the sub-sample with laboratory values was applied to reduce bias and approximate a nationally, representative sample. Arterial stiffness was assessed by arterial pulse pressure (APP). Results APP was higher for MA [B = 0.063 (95% CI 0.015 to 0.111), p = 0.013] and BNH [B = 0.044 (95% CI 0.006 to 0.082), p = 0.018] than WNH, controlling for diabetes, age, gender, body mass index (BMI), fiber intake, energy intake (Kcal) and smoking. A two-way interaction of diabetes by carbohydrate intake (grams) was inversely associated with APP [B = -1.18 (95% CI -0.178 to -0.058), p = 0.001], controlling for race, age, gender, BMI, Kcal and smoking. BNH with diabetes who consumed more mono-unsaturated fatty acids (MUFA) than WNH with diabetes had lower APP [B = -0.112 (95%CI-0.179 to -0.045), p = 0.003] adjusting for saturated fatty acids, Kcal, age, gender, BMI and smoking. Conclusion Higher MUFA and carbohydrate intake for persons with diabetes reflecting lower APP may be due to replacement of saturated fats with CHO and MUFA. The associations of APP with diabetes, race and dietary intake need to be confirmed with intervention and prospective studies. Confirmation of these results would suggest that dietary interventions for minorities with diabetes may improve arterial health.

Identification of Anziaic Acid, a Lichen Depside from Hypotrachyna sp., as a New Topoisomerase Poison Inhibitor

Topoisomerase inhibitors are effective for antibacterial and anticancer therapy because they can lead to the accumulation of the intermediate DNA cleavage complex formed by the topoisomerase enzymes, which trigger cell death. Here we report the application of a novel enzyme-based high-throughput screening assay to identify natural product extracts that can lead to increased accumulation of the DNA cleavage complex formed by recombinant Yersinia pestistopoisomerase I as part of a larger effort to identify new antibacterial compounds. Further characterization and fractionation of the screening positives from the primary assay led to the discovery of a depside, anziaic acid, from the lichen Hypotrachyna sp. as an inhibitor for both Y. pestis and Escherichia colitopoisomerase I. In in vitro assays, anziaic acid exhibits antibacterial activity against Bacillus subtilis and a membrane permeable strain of E. coli. Anziaic acid was also found to act as an inhibitor of human topoisomerase II but had little effect on human topoisomerase I. This is the first report of a depside with activity as a topoisomerase poison inhibitor and demonstrates the potential of this class of natural products as a source for new antibacterial and anticancer compounds.

Community control in cellular protein production : consequences for amino acid starvation

The authors thank M. C. Romano, I. Stansfield, L. Ciandrini, A. Kort, and M. Rehberg for helpful discussions. This work was funded by BBSRC grants BB/F00513/X1 and BB/G010722, and the Scottish Universities Life Science Alliance (SULSA).

Community control in cellular protein production : consequences for amino acid starvation

The authors thank M. C. Romano, I. Stansfield, L. Ciandrini, A. Kort, and M. Rehberg for helpful discussions. This work was funded by BBSRC grants BB/F00513/X1 and BB/G010722, and the Scottish Universities Life Science Alliance (SULSA).

Programming Molecular Devices using Nucleic Acid Hairpins

Nucleic Acid hairpins have been a subject of study for the last four decades. They are composed of single strand that is

hybridized to itself, and the central section forming an unhybridized loop. In nature, they stabilize single stranded RNA, serve as nucleation

sites for RNA folding, protein recognition signals, mRNA localization and regulation of mRNA degradation. On the other hand,

DNA hairpins in biological contexts have been studied with respect to forming cruciform structures that can regulate gene expression.

The use of DNA hairpins as fuel for synthetic molecular devices, including locomotion, was proposed and experimental demonstrated in 2003. They

were interesting because they bring to the table an on-demand energy/information supply mechanism.

The energy/information is hidden (from hybridization) in the hairpin’s loop, until required.

The energy/information is harnessed by opening the stem region, and exposing the single stranded loop section.

The loop region is now free for possible hybridization and help move the system into a thermodynamically favourable state.

The hidden energy and information coupled with

programmability provides another functionality, of selectively choosing what reactions to hide and

what reactions to allow to proceed, that helps develop a topological sequence of events.

Hairpins have been utilized as a source of fuel for many different DNA devices. In this thesis, we program four different

molecular devices using DNA hairpins, and experimentally validate them in the

laboratory. 1) The first device: A

novel enzyme-free autocatalytic self-replicating system composed entirely of DNA that operates isothermally. 2) The second

device: Time-Responsive Circuits using DNA have two properties: a) asynchronous: the final output is always correct

regardless of differences in the arrival time of different inputs.

b) renewable circuits which can be used multiple times without major degradation of the gate motifs

(so if the inputs change over time, the DNA-based circuit can re-compute the output correctly based on the new inputs).

3) The third device: Activatable tiles are a theoretical extension to the Tile assembly model that enhances

its robustness by protecting the sticky sides of tiles until a tile is partially incorporated into a growing assembly.

4) The fourth device: Controlled Amplification of DNA catalytic system: a device such that the amplification

of the system does not run uncontrollably until the system runs out of fuel, but instead achieves a finite

amount of gain.

Nucleic acid circuits with the ability

to perform complex logic operations have many potential practical applications, for example the ability to achieve point of care diagnostics.

We discuss the designs of our DNA Hairpin molecular devices, the results we have obtained, and the challenges we have overcome

to make these truly functional.

(Table 1) Average sea surface temperature, average sea surface salinity, C37 concentration, palmitic acid concentration, Uk'37, C37/C38 ratio, dD of water, dD of C37 and dD of palmitic acid of water samples from a transect across the Amazon Plume

The stable hydrogen isotope composition of lipid biomarkers, such as alkenones, is a promising new tool for the improvement of palaeosalinity reconstructions. Laboratory studies confirmed the correlation between lipid biomarker dD composition (dDLipid), water dD composition (dDH2O) and salinity; yet there is limited insight into the applicability of this proxy in oceanic environments. To fill this gap, we test the use of the dD composition of alkenones (dDC37) and palmitic acid (dDPA) as salinity proxies using samples of surface suspended material along the distinct salinity gradient induced by the Amazon Plume. Our results indicate a positive correlation between salinity and dDH2O, while the relationship between dDH2O and dDLipid is more complex: dDPAM correlates strongly with dDH2O (r2 = 0.81) and shows a salinity-dependent isotopic fractionation factor. dDC37 only correlates with dDH2O in a small number (n = 8) of samples with alkenone concentrations > 10 ng L**-1, while there is no correlation if all samples are taken into account. These findings are mirrored by alkenone-based temperature reconstructions, which are inaccurate for samples with low alkenone concentrations. Deviations in dDC37 and temperature are likely to be caused by limited haptophyte algae growth due to low salinity and light limitation imposed by the Amazon Plume. Our study confirms the applicability of dDLipid as a salinity proxy in oceanic environments. But it raises a note of caution concerning regions where low alkenone production can be expected due to low salinity and light limitation, for instance, under strong riverine discharge.

Differential susceptibility of mitochondrial complex II to inhibition by oxaloacetate in brain and heart

Mitochondrial Complex II is a key mitochondrial enzyme connecting the tricarboxylic acid (TCA) cycle and the electron transport chain. Studies of complex II are clinically important since new roles for this enzyme have recently emerged in cell signalling, cancer biology, immune response and neurodegeneration. Oxaloacetate (OAA) is an intermediate of the TCA cycle and at the same time is an inhibitor of complex II with high affinity (Kd ~ 10− 8 M). Whether or not OAA inhibition of complex II is a physiologically relevant process is a significant, but still controversial topic. We found that complex II from mouse heart and brain tissue has similar affinity to OAA and that only a fraction of the enzyme in isolated mitochondrial membranes (30.2 ± 6.0% and 56.4 ± 5.6% in the heart and brain, respectively) is in the free, active form. Since OAA could bind to complex II during isolation, we established a novel approach to deplete OAA in the homogenates at the early stages of isolation. In heart, this treatment significantly increased the fraction of free enzyme, indicating that OAA binds to complex II during isolation. In brain the OAA-depleting system did not significantly change the amount of free enzyme, indicating that a large fraction of complex II is already in the OAA-bound inactive form. Furthermore, short-term ischemia resulted in a dramatic decline of OAA in tissues, but it did not change the amount of free complex II. Our data show that in brain OAA is an endogenous effector of complex II, potentially capable of modulating the activity of the enzyme.

Direct catalytic macrolactonization and application of the phase separation strategy in complex molecule synthesis

Les macrolactones sont des squelettes structuraux importants dans de nombreuses sphères de l’industrie chimique, en particulier dans les marchés pharmaceutiques et cosmétiques. Toutefois, la stratégie traditionnelle pour la préparation de macrolactones demeure incommode en requérant notamment l’ajout (super)stœchiométrique d’agents activateurs. Conséquemment, des quantités stœchiométriques de sous-produits sont générées; ils sont souvent toxiques, dommageables pour l’environnement et nécessitent des méthodes de purification fastidieuses afin de les éliminer. La présente thèse décrit le développement d’une macrolactonisation efficace catalysée au hafnium directement à partir de précurseurs portant un acide carboxylique et un alcool primaire, ne générant que de l’eau comme sous-produit et ne nécessitant pas de techniques d’addition lente et/ou azéotropique. Le protocole a également été adapté à la synthèse directe de macrodiolides à partir de mélanges équimolaires de diols et de diacides carboxyliques et à la synthèse de dimères tête-à-queue de seco acides. Des muscs macrocycliques ainsi que des macrolactones pertinentes à la chimie médicinale ont pu être synthétisés avec l’approche développée. Un protocole pour l’estérification directe catalysée au hafnium entre des acides carboxyliques et des alcools primaires a aussi été développé. Différentes méthodes pour la macrolactonisation catalytique directe entre des alcools secondaires et des acides carboxyliques ont été étudiées. En outre, la stratégie de séparation de phase en macrocyclisation en débit continu a été appliquée lors de la synthèse totale formelle de la macrolactone ivorenolide A. Les étapes-clés de la synthèse incluent une macrocyclisation par le couplage d’alcynes de Glaser-Hay et une réaction de métathèse d’alcènes Z-sélective.

Nucleic Acid Diagnostics using Isotachophoresis and Isothermal Amplification

Thesis (Ph.D.)--University of Washington, 2016-08

Transcriptome, carbohydrate, and phytohormone analysis of Petunia hybrida reveals a complex disturbance of plant functional integrity under mild chilling stress

Cultivation of chilling-tolerant ornamental crops at lower temperature could reduce the energy demands of heated greenhouses. To provide a better understanding of how sub-optimal temperatures (12 degrees C vs. 16 degrees C) affect growth of the sensitive Petunia hybrida cultivar 'SweetSunshine Williams', the transcriptome, carbohydrate metabolism, and phytohormone homeostasis were monitored in aerial plant parts over 4 weeks by use of a microarray, enzymatic assays and GC-MS/MS. The data revealed three consecutive phases of chilling response. The first days were marked by a strong accumulation of sugars, particularly in source leaves, preferential up-regulation of genes in the same tissue and down-regulation of several genes in the shoot apex, especially those involved in the abiotic stress response. The midterm phase featured a partial normalization of carbohydrate levels and gene expression. After 3 weeks of chilling exposure, a new stabilized balance was established. Reduced hexose levels in the shoot apex, reduced ratios of sugar levels between the apex and source leaves and a higher apical sucrose/hexose ratio, associated with decreased activity and expression of cell wall invertase, indicate that prolonged chilling induced sugar accumulation in source leaves at the expense of reduced sugar transport to and reduced sucrose utilization in the shoot. This was associated with reduced levels of indole-3-acetic acid and abscisic acid in the apex and high numbers of differentially, particularly up-regulated genes, especially in the source leaves, including those regulating histones, ethylene action, transcription factors, and a jasmonate-ZIM-domain protein. Transcripts of one Jumonji C domain containing protein and one expansin accumulated in source leaves throughout the chilling period. The results reveal a dynamic and complex disturbance of plant function in response to mild chilling, opening new perspectives for the comparative analysis of differently tolerant cultivars.

Life without complex I: proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase complex

The mitochondrial NADH dehydrogenase complex (complex I) is of particular importance for the respiratory chain in mitochondria. It is the major electron entry site for the mitochondrial electron transport chain (mETC) and therefore of great significance for mitochondrial ATP generation. We recently described an Arabidopsis thaliana double-mutant lacking the genes encoding the carbonic anhydrases CA1 and CA2, which both form part of a plant-specific 'carbonic anhydrase domain' of mitochondrial complex I. The mutant lacks complex I completely. Here we report extended analyses for systematically characterizing the proteome of the ca1ca2 mutant. Using various proteomic tools, we show that lack of complex I causes reorganization of the cellular respiration system. Reduced electron entry into the respiratory chain at the first segment of the mETC leads to induction of complexes II and IV as well as alternative oxidase. Increased electron entry at later segments of the mETC requires an increase in oxidation of organic substrates. This is reflected by higher abundance of proteins involved in glycolysis, the tricarboxylic acid cycle and branched-chain amino acid catabolism. Proteins involved in the light reaction of photosynthesis, the Calvin cycle, tetrapyrrole biosynthesis, and photorespiration are clearly reduced, contributing to the significant delay in growth and development of the double-mutant. Finally, enzymes involved in defense against reactive oxygen species and stress symptoms are much induced. These together with previously reported insights into the function of plant complex I, which were obtained by analysing other complex I mutants, are integrated in order to comprehensively describe 'life without complex I'.

Direct catalytic macrolactonization and application of the phase separation strategy in complex molecule synthesis

Les macrolactones sont des squelettes structuraux importants dans de nombreuses sphères de l’industrie chimique, en particulier dans les marchés pharmaceutiques et cosmétiques. Toutefois, la stratégie traditionnelle pour la préparation de macrolactones demeure incommode en requérant notamment l’ajout (super)stœchiométrique d’agents activateurs. Conséquemment, des quantités stœchiométriques de sous-produits sont générées; ils sont souvent toxiques, dommageables pour l’environnement et nécessitent des méthodes de purification fastidieuses afin de les éliminer. La présente thèse décrit le développement d’une macrolactonisation efficace catalysée au hafnium directement à partir de précurseurs portant un acide carboxylique et un alcool primaire, ne générant que de l’eau comme sous-produit et ne nécessitant pas de techniques d’addition lente et/ou azéotropique. Le protocole a également été adapté à la synthèse directe de macrodiolides à partir de mélanges équimolaires de diols et de diacides carboxyliques et à la synthèse de dimères tête-à-queue de seco acides. Des muscs macrocycliques ainsi que des macrolactones pertinentes à la chimie médicinale ont pu être synthétisés avec l’approche développée. Un protocole pour l’estérification directe catalysée au hafnium entre des acides carboxyliques et des alcools primaires a aussi été développé. Différentes méthodes pour la macrolactonisation catalytique directe entre des alcools secondaires et des acides carboxyliques ont été étudiées. En outre, la stratégie de séparation de phase en macrocyclisation en débit continu a été appliquée lors de la synthèse totale formelle de la macrolactone ivorenolide A. Les étapes-clés de la synthèse incluent une macrocyclisation par le couplage d’alcynes de Glaser-Hay et une réaction de métathèse d’alcènes Z-sélective.

Bioorthogonal Noncanonical Amino Acid Tagging for Selective Analysis of the Pseudomonas aeruginosa Proteome

In natural environments, bacterial physiology is frequently characterized by slow metabolic rates and complex cellular heterogeneities. The opportunistic pathogen Pseudomonas aeruginosa provides one such example; P. aeruginosa forms untreatable chronic biofilm infections of the cystic fibrosis lung, where oxygen limitation can lead to states of metabolic dormancy. To better understand the biology of these states, in vitro experiments must be adapted to better recapitulate natural settings. However, low rates of protein turnover and cellular or phenotypic complexity make these systems difficult to study using established methods. Here we adapt the bioorthogonal noncanonical amino acid tagging (BONCAT) method for time- and cell-selective proteomic analysis to the study of P. aeruginosa. Analysis of proteins synthesized in an anoxic dormancy state led to the discovery of a new type of transcriptional regulator which we designated SutA. We performed detailed analyses of SutA’s role in transcription under slow growth states and we elucidated the structural basis for its regulatory behavior. Additionally, we used cell-selective targeting of BONCAT labeling to measure the dynamic proteomic response of an antibiotic-tolerant biofilm subpopulation. Overall this work shows the utility of selective proteomics as applied to bacterial physiology and describes the broad biological insight obtained from that application.

Transport in the blood of an anti-tumor water-soluble rutheniun cyclopentadienyl complex: a fluorescence study on albumin binding

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Development of two charge transfer complex spectrophotometric methods for determination of tofisopam in tablet dosage form

Purpose: To develop a simple, fast and sensitive spectrophotometric method for the determination of tofisopam in tablet dosage form. Methods: Tofisopam as n-electron donor was reacted with two π-acceptors, namely, chloranilic acid (ChA), and 7,7,8,8 tetracyanoquinodimethane (TCNQ) to form charge transfer complexes. The complexes were evaluated spectrophotometrically at 520 and 824 nm for ChA and TCNQ, respectively. The optimum conditions for the reaction were determined and optimized. The developed method was compared with Japanese Pharmacopeia method. Results: The calibration curve was linear in the ranges 25 – 125 and 30 – 150 μg/mL for ChA and TCNQ, respectively. The lower limit of detection was 8.0 and 10.0 μg/mL for ChA and TCNQ, respectively while the slope and intercept of the calibration curves were 0.0025 and 0.011 and 0.0115 and -0.237, for ChA and TCNQ, respectively. Conclusion: The developed methods for tofisopam have good accuracy and precision, and comparable to a standard pharmacopeial method. The methods can be applied for routine analysis and in quality control.