The presence of a non-B-Z-structure enhances an enzyme's reactivity at its recognition site

Alternating (CG) sequences form an unusual conformation in the presence of cobalt hexamine. The oligomer, BZ-IV, containing a (CG)4 run (BZ-IV sequence: 5'TCGACGCGCGCGATCAGTCA- 3') was inserted at the Sal I site of the Escherichia coli pGEM-5zf(+) plasmid producing the plasmid pCW001. Hinf I digestion of pCW001 produced a 367 base pair (bp) fragment containing the BZ-IV insert. For controls, the 452 bp Hinf I fragment from the pCW001 plasmid and the 347 bp Hinf I fragment from the pGEM plasmid were used. Digestion studies were performed using the restriction enzymes Bgl I, EcoRV, Hha I, Mbo I, Not I, Pst I, and Taq I and methylation studies were performed using dam methylase. Data were obtained by beta scanning or ethidium bromide staining the polyacrylamide gels of the digestion or methylation products. The results show that in the presence of 100 uM cobalt hexamine, in which BZ-IV takes on a non-B-Z-structure, the enzyme's ability to react and cleave its recognition site is enhanced.

Reactions of singlet oxygen and singlet oxygen mimics with dicyclopropylethylenes: comparison of reactivity and mechanisms

An extensive study of the reaction pathways of 1,1- dicyclopropyl ethylene, cis- and trans- 1,2-dicyclopropylethylenes has been undertaken with different electrophiles 4-methyl-1,2,4- triazoline-3,5-dione (MTAD), tetracyanoethylene (TCNE), and singlet oxygen (102). Comparison of reactivity and reaction mechanisms among the electrophiles is investigated. Singlet oxygen exhibits significantly lower reactivity compared to the other electrophiles. MTAD and TCNE react with dicyclopropylethylenes to produce predominantly "2+2" adducts and a small amount of the "ene" adducts. The "2+2" is the major product presumably because of the high activation energy leading to the highly strained "ene" products. Solvent trapping studies provide strong evidence of a "stepwise" mechanism, involving a zwitterionic or aziridinium imide as an intermediate from the study of the reactions products of dicyclopropylethylenes and MTAD.

Sasha Wagner collects surface water samples to assess dissolved organic matter sources and reactivity along the Harney River

http://digitalcommons.fiu.edu/fce_lter_photos/1302/thumbnail.jpg

Validity of the portable piko-6 spirometer used for screening obstructive airway diseases in community pharmacy practice

Background: Obstructive airway diseases (OADs) are among the leading causes of morbidity and mortality worldwide. Shortness of breath (SOB) is the main symptom associated with OADs. International guidelines from the Global Initiative for Chronic Lung Disease (GOLD) and the Global Initiative for Asthma (GINA) have recommended spirometry as an indispensable tool for the diagnosis of asthma and chronic obstructive pulmonary diseases (COPD), but spirometry is rarely used in family practice. Simple and reliable diagnostic tools are necessary for screening community patients with onset of OADs for timely management. Purpose: This thesis examined screening utility of the PiKo-6 forced expiratory volume in one second (pFEV₁) , in six second (pFEV₆), and the pRatio ( pFEV₁/pFEV₆) in SOB patients for OADs in community pharmacy settings. FEV₆ has recently been suggested an excellent surrogate for Forced Vital Capacity (FVC), which requires maximum exhalation of the lungs. Methods: Patients with SOB symptoms who were prescribed pulmonary inhalers, by their family physicians, were recruited via community pharmacies. Trained pharmacists collected two PiKo-6 tests to assess the repeatability of the PiKo-6 device. All patients performed laboratory spirometry ( FEV₁, FVC and FEV₁/FVC) to obtain physician diagnosis of their OADs. The results of the PiKo-6 spirometer and laboratory spirometer were compared. In addition, the PiKo-6 pRatio and laboratory FEV₁/FVC were assessed against physician diagnosed COPD. Results: Sixty three patients volunteered to perform the PiKo-6 spirometry. Of these, 52.4 % were men (age 53.9 ± 15.3 years; BMI 31.9 ± 7.40 kg/m2). Repeated testing with pFEV₁, pFEV6 and pRatio correlated significantly (within correlation, r = 0.835, p-Value≤ 0.05 ; 0.872, p- Value≤ 0.05; and 0.664, p-Value≤ 0.05). In addition, pFEV₁, pFEV6 and pRatio correlated significantly with FEV₁, FVC and FEV₁/FVC, respectively (between correlation = 0.630, p- Value≤ 0.05 ; 0.660, p-Value≤ 0.05 and 0.580, p-Value≤ 0.05). The cut-off value corresponding to the greatest sum of sensitivity and specificity of pRatio for physician-diagnosed COPD was <0.80, the sensitivity and specificity were 84 % and 50%, respectively. Conclusions The portable PiKo-6 correlates moderately well with the standard spirometry, when delivered by community pharmacists to patients with OADs. The PiKo-6 spirometer may play a role in screening patients suspected of having an OAD in community pharmacies that may benefit from early physician diagnosis and appropriate management.

Late Transition Metal-Oxo complexes: Synthesis, and Biorelevant Reactivity

High-valent terminal metal-oxygen adducts are supposed to be potent oxidising intermediates in enzymatic catalyses. In contrast to those from groups 6-8, oxidants that contain late transition metals (Co, Ni, Cu) are poorly understood. Because of their high reactivity, only a few examples of these compounds have been observed. The aim of this project was to investigate the reactivity of high-valent Ni(III) complexes, containing a monodentate oxygen-donor ligands, in hydrogen atom abstraction (HAA) and oxygen atom transfer (OAT) reactions which are typical of biological high-valent metal-oxygen species. Particularly, the Ni(III) complexes were generated in situ, at low temperature, from the oxidation of the Ni(II) species.The nickel complexes studied during this work were supported by tridentate ligands, with a strong σ-donating ability and exceedingly resistant to several common degradation pathways. These complexes vary based on the monodentate group in the fourth coordination position site, which can be neutral or anionic. In particular, we prepared four different Ni(III) complexes [NiIII(pyN2Me2)(OCO2H)] (12), [NiIII(pyN2Me2)(ONO2)] (14), [NiIII(pyN2Me2)(OC(O)CH3)] (18) and [NiIII(pyN2Me2)(OC(O)H)] (25). They feature a bicarbonate (-OCO2H), nitrate (-ONO2), acetate (-OC(O)CH3) and formate (-OC(O)H) group, respectively.HAA and OAT reactions were performed by adding 2,6-di-tert-butylphenol (2,6-DTBP) at -40°C, and triphenylphosphine (PPh3) at -80°C, to the in situ generated Ni(III) complexes, respectively. These reactions were carried out by adding 7 to 500 equivalents of substrate, in order to ensure pseudo-first order conditions. Since, the reactivity of the Ni(III) complex featured by the bicarbonate group has been studied in a previous work, we only investigated that of the species bearing the nitrate, acetate and formate ligand. Finally we compared the value of the reaction rate of all the four species in the HAA and OAT reactions.

Hg speciation in petroleum hydrocarbons with emphasis on the reactivity of Hg particles

Peer reviewed

Molecular Mechanisms of Airway Epithelial Progenitor Cell Maintenance and Repair.

The lungs are vital organs whose airways are lined with a continuous layer of epithelial cells. Epithelial cells in the distal most part of the lung, the alveolar space, are specialized to facilitate gas exchange. Proximal to the alveoli is the airway epithelium, which provides an essential barrier and is the first line of defense against inhaled toxicants, pollutants, and pathogens. Although the postnatal lung is a quiescent organ, it has an inherent ability to regenerate in response to injury. Proper balance between maintaining quiescence and undergoing repair is crucial, with imbalances in these processes leading to fibrosis or tumor development. Stem and progenitor cells are central to maintaining balance, given that they proliferate and renew both themselves and the various differentiated cells of the lung. However, the precise mechanisms regulating quiescence and repair in the lungs are largely unknown. In this dissertation, ionizing radiation is used as a physiologically relevant injury model to better understand the repair process of the airway epithelium. We use in vitro and in vivo mouse models to study the response of a secretory progenitor, the club cell, to various doses and qualities of ionizing radiation. Exposure to radiation found in space environments and in some types of radiotherapy caused clonal expansion of club cells specifically in the most distal branches of the airway epithelium, indicating that the progenitors residing in the terminal bronchioles are radiosensitive. This clonal expansion is due to an increase in p53-dependent apoptosis, senescence, and mitotic defects. Through the course of this work, we discovered that p53 is not only involved in radiation response, but is also a novel regulator of airway epithelial homeostasis. p53 acts in a gene dose-dependent manner to regulate the composition of airway epithelium by maintaining quiescence and regulating differentiation of club progenitor cells in the steady-state lung. The work presented in this dissertation represents an advance in our understanding of the molecular mechanisms underlying maintenance of airway epithelial progenitor cells as well as their repair following ionizing radiation exposure.

When static meets dynamic: Comparing cone-beam computed tomography and acoustic reflection for upper airway analysis

INTRODUCTION: Upper airway measurement can be important for the diagnosis of breathing disorders. Acoustic reflection (AR) is an accepted tool for studying the airway. Our objective was to investigate the differences between cone-beam computed tomography (CBCT) and AR in calculating airway volumes and areas. METHODS: Subjects with prescribed CBCT images as part of their records were also asked to have AR performed. A total of 59 subjects (mean age, 15 ± 3.8 years) had their upper airway (5 areas) measured from CBCT images, acoustic rhinometry, and acoustic pharyngometry. Volumes and minimal cross-sectional areas were extracted and compared with software. RESULTS: Intraclass correlation on 20 randomly selected subjects, remeasured 2 weeks apart, showed high reliability (r >0.77). Means of total nasal volume were significantly different between the 2 methods (P = 0.035), but anterior nasal volume and minimal cross-sectional area showed no differences (P = 0.532 and P = 0.066, respectively). Pharyngeal volume showed significant differences (P = 0.01) with high correlation (r = 0.755), whereas pharyngeal minimal cross-sectional area showed no differences (P = 0.109). The pharyngeal volume difference may not be considered clinically significant, since it is 758 mm3 for measurements showing means of 11,000 ± 4000 mm3. CONCLUSIONS: CBCT is an accurate method for measuring anterior nasal volume, nasal minimal cross-sectional area, pharyngeal volume, and pharyngeal minimal cross-sectional area.

In Vitro modelling of RSV infection and cytopathogenesis in well-differentiated human primary airway epithelial cells (WD-PAECs).

The choice of model used to study human respiratory syncytial virus (RSV) infection is extremely important. RSV is a human pathogen that is exquisitely adapted to infection of human hosts. Rodent models, such as mice and cotton rats, are semi-permissive to RSV infection and do not faithfully reproduce hallmarks of RSV disease in humans. Furthermore, immortalized airway-derived cell lines, such as HEp-2, BEAS-2B, and A549 cells, are poorly representative of the complexity of the respiratory epithelium. The development of a well-differentiated primary pediatric airway epithelial cell models (WD-PAECs) allows us to simulate several hallmarks of RSV infection of infant airways. They therefore represent important additions to RSV pathogenesis modeling in human-relevant tissues. The following protocols describe how to culture and differentiate both bronchial and nasal primary pediatric airway epithelial cells and how to use these cultures to study RSV cytopathogenesis.

A Selective Irreversible Inhibitor of Furin Does Not Prevent Pseudomonas Aeruginosa Exotoxin A-Induced Airway Epithelial Cytotoxicity

Many bacterial and viral pathogens (or their toxins), including Pseudomonas aeruginosa exotoxin A, require processing by host pro-protein convertases such as furin to cause dis- ease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consist- ing of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar com- pound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent P. aerugi- nosa exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process.

The role of anaerobic bacteria in the cystic fibrosis airway

PURPOSE OF REVIEW: Anaerobic bacteria are not only normal commensals, but are also considered opportunistic pathogens and have been identified as persistent members of the lower airway community in people with cystic fibrosis of all ages and stages of disease. Currently, the role of anaerobic bacteria in cystic fibrosis lower airway disease is not well understood. Therefore, this review describes the recent studies relating to the potential pathophysiological role(s) of anaerobes within the cystic fibrosis lungs.

RECENT FINDINGS: The most frequently identified anaerobic bacteria in the lower airways are common to both cystic fibrosis and healthy lungs. Studies have shown that in cystic fibrosis, the relative abundance of anaerobes fluctuates in the lower airways with reduced lung function and increased inflammation associated with a decreased anaerobic load. However, anaerobes found within the lower airways also produce virulence factors, may cause a host inflammatory response and interact synergistically with recognized pathogens.

SUMMARY: Anaerobic bacteria are potentially members of the airway microbiota in health but could also contribute to the pathogenesis of lower airway disease in cystic fibrosis via both direct and indirect mechanisms. A personalized treatment strategy that maintains a normal microbial community may be possible in the future.

Effect of heat treatment on reactivity-strength of alkali-activated natural pozzolans

Natural pozzolans can be activated and condensed with sodium silicate in an alkaline environment to synthesize high performance cementitious construction materials with low environmental impact. The nature of the starting materials including mineral composition, chemical composition and crystal structure groups affects the formation of the geopolymer gel phase. In this paper, the pozzolanic activities of five natural pozzolans are studied. From XRD and XRF results, most of the raw materials contain zeolite clay minerals and have a high loss on ignition. Therefore, before use, samples were calcined at 700, 800 and 900 °C, respectively. The improvement in pozzolanic properties was studied following heat treatment including calcinations and/or elevated curing temperature by using alkali solubility and compressive strength tests. The results show that pozzolan containing sodium zeolite clinoptilolite can be used to prepare a moderate to high strength binder by heat treatment and calcinations can impart disorder hornblende as a constituent of pozzolan with no amorphous phase to prepare a moderate strength binder.

Catalytic Processes Mediated by Metal−Organic Frameworks : Reactivity and Mechanistic Studies

The present thesis describes the development of heterogeneous catalytic methodologies using metal−organic frameworks (MOFs) as porous matrices for supporting transition metal catalysts. A wide spectrum of chemical reactions is covered. Following the introductory section (Chapter 1), the results are divided between one descriptive part (Chapter 2) and four experimental parts (Chapters 3–6). Chapter 2 provides a detailed account of MOFs and their role in heterogeneous catalysis. Specific synthesis methods and characterization techniques that may be unfamiliar to organic chemists are illustrated based on examples from this work. Pd-catalyzed heterogeneous C−C coupling and C−H functionalization reactions are studied in Chapter 3, with focus on their practical utility. A vast functional group tolerance is reported, allowing access to substrates of relevance for the pharmaceutical industry. Issues concerning the recyclability of MOF-supported catalysts, leaching and operation under continuous flow are discussed in detail. The following chapter explores puzzling questions regarding the nature of the catalytically active species and the pathways of deactivation for Pd@MOF catalysts. These questions are addressed through detailed mechanistic investigations which include in situ XRD and XAS data acquisition. For this purpose a custom reaction cell is also described in Chapter 4. The scope of Pd@MOF-catalyzed reactions is expanded in Chapter 5. A strategy for boosting the thermal and chemical robustness of MOF crystals is presented. Pd@MOF catalysts are coated with a protecting SiO2 layer, which improves their mechanical properties without impeding diffusion. The resulting nanocomposite is better suited to withstand the harsh conditions of aerobic oxidation reactions. In this chapter, the influence of the nanoparticles’ geometry over the catalyst’s selectivity is also investigated. While Chapters 3–5 dealt with Pd-catalyzed processes, Chapter 6 introduces hybrid materials based on first-row transition metals. Their reactivity is explored towards light-driven water splitting. The heterogenization process leads to stabilized active sites, facilitating the spectroscopic probing of intermediates in the catalytic cycle.