Structural Basis of RNA Recognition by Mycobacterium tuberculosis MazF Homologues

The MazEF toxin-antitoxin (TA) system consists of the antitoxin MazE and the toxin MazF. MazF is a sequence-specific endoribonuclease that upon activation causes cellular growth arrest and increass the level of persisters. Moreover, MazF-induced cells are in a quasi-dormant state that cells remain metabolically active while stop dividing. The quasi-dormancy is similar to the nonreplicating state of M. tuberculosis during latent tuberculosis, thus suggesting the role of mazEF in M. tuberculosis dormancy and persistence. M. tuberculosis has nine mazEF TA modules, each with different RNA cleavage specificities and implicated in selective gene expression during stress conditions. To date only the Bacillus subtilis MazF-RNA complex structure has been determined. As M. tuberculosis MazF homologues recognize distinct RNA sequences, their molecular mechanisms of substrate specificity remain unclear. By taking advantage of X-ray crystallography, we have determined structures of two M. tuberculosis MazF-RNA complexes, MazF-mt1 (Rv2801c) and MazF-mt3 (Rv1991c) in complex with an uncleavable RNA substrate. These structures have provided the molecular basis of sequence-specific RNA recognition and cleavage by MazF toxins.

Both MazF-mt1-RNA and MazF-mt3-RNA complexes showed similar structural organization with one molecule of RNA bound to a MazF-mt1 or MazF-mt3 dimer and occupying the same pocket within the MazF dimer interface. Similar to B. subtilis MazF-RNA complex, MazF-mt1 and MazF-mt3 displayed a conserved active site architecture, where two highly conserved residues, Arg and Thr, form hydrogen bonds with the scissile phosphate group in the cleavage site of the bound RNA. The MazF-mt1-RNA complex also showed specific interactions with its three-base RNA recognition element. Compared with the B. subtilis MazF-RNA complex, our structures showed that residues involved in sequence-specific recognition of target RNA vary between the MazF homologues, therefore explaining the molecular basis for their different RNA recognition sequences. In addition, local conformational changes of the loops in the RNA binding site of MazF-mt1 appear to play a role in MazF targeting different RNA lengths and sequences. In contrast, the MazF-mt3-RNA complex is in a non-optimal RNA binding state with a symmetry-related MazF-mt3 molecule found to make interactions with the bound RNA in the crystal. The crystal-packing interactions were further examined by isothermal titration calorimetry (ITC) studies on selected MazF-mt3 mutants. Our attempts to utilize a MazF-mt3 mutant bearing mutations involved in crystal contacts all crystallized with few nucleotides, which are still found to interact with a symmetry mate. However, these different crystal forms revealed the conformational flexibility of loops in the RNA binding interface of MazF-mt3, suggesting their role in RNA binding and recognition, which will require further studies on additional MazF-mt3-RNA complex interactions.

In conclusion, the structures of the MazF-mt1-RNA and MazF-mt3-RNA complexes provide the first structural information on any M. tuberculosis MazF homologues. Supplemented with structure-guided mutational studies on MazF toxicity in vivo, this study has addressed the structural basis of different RNA cleavage specificities among MazF homologues. Our work will guide future studies on the function of other M. tuberculosis MazF and MazE-MazF homologues, and will help delineate their physiological roles in M. tuberculosis stress responses and pathogenesis.

A Loop-mediated Isothermal Amplification Method for Rapid Direct Detection and Differentiation of Non-pathogenic and Verocytotoxigenic Escherichia coli in Beef and Bovine Faeces

The aim of this study was to develop a multiplex loop-mediated isothermal amplification (LAMP) method capable of detecting Escherichia coli generally and verocytotoxigenic E. coli (VTEC) specifically in beef and bovine faeces. The LAMP assay developed was highly specific (100%) and able to distinguish between E. coli and VTEC based on the amplification of the phoA, and stx1 and/or stx2 genes, respectively. In the absence of an enrichment step, the limit of detection 50% (LOD50) of the LAMP assay was determined to be 2.83, 3.17 and 2.83-3.17 log CFU/g for E. coli with phoA, stx1 and stx2 genes, respectively, when artificially inoculated minced beef and bovine faeces were tested. The LAMP calibration curves generated with pure cultures, and spiked beef and faeces, suggested that the assay had good quantification capability. Validation of the assay, performed using retail beef and bovine faeces samples, demonstrated good correlation between counts obtained by the LAMP assay and by a conventional culture method, but suggested the possibility of false negative LAMP results for 12.5-14.7% of samples tested. The multiplex LAMP assay developed potentially represents a rapid alternative to culture for monitoring E.coli levels in beef or faeces and it would provide additional information on the presence of VTEC. However, some further optimisation is needed to improve detection sensitivity.

Nucleic Acid Diagnostics using Isotachophoresis and Isothermal Amplification

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

Adsorption Calorimetry for Energy Conversion Technologies: Applications in Organic Photovoltaics, Catalysis, and Atomic Layer Deposition

Thesis (Ph.D.)--University of Washington, 2016-07

EFFECT OF ISOTHERMAL AGING ON SAC305 HARMONIC VIBRATION DURABILITY

The effect of isothermal aging on the harmonic vibration durability of Sn3.0Ag0.5Cu solder interconnects is examined. Printed wiring assemblies with daisy-chained leadless chip resistors (LCRs) are aged at 125°C for 0, 100, and 500 hours. These assemblies are instrumented with accelerometers and strain gages to maintain the same harmonic vibration profile in-test, and to characterize PWB behavior. The tested assemblies are excited at their first natural frequencies until LCRs show a resistance increase of 20%. Dynamic finite element models are employed to generate strain transfer functions, which relate board strain levels observed in-test to respective solder strain levels. The transfer functions are based on locally averaged values of strains in critical regions of the solder and in appropriate regions of the PWB. The vibration test data and the solder strains from FEA are used to estimate lower-bound material fatigue curves for SAC305 solder materials, as a function of isothermal pre-aging.

Sequestering of Fe and Pb ions from Wastewater by Canarium schweinfurthii after carbonization and chemical treatment: Isothermal and Kinetic Modeling

In this paper agricultural waste; Canarium schweinfurthii was explored for the sequestering of Fe and Pb ions from wastewater solution after carbonization and chemical treatment at 400oC. Optimum time of 30 and 150 min with percentage removal of 95 and 98% at optimum pH of 2 and 6 was obtained for Fe and Pb ions. Kinetics model followed pseudofirst order as sum of absolute error (EABS) between Qe and Qc greater than that of pseudo second order. Parameters evaluated from isothermal equation (Freundlich and Langmuir) showed that KL and QO for Fe > Pb and R2 for Langmuir> Freundlich. The study reveals the suitability of the adsorbent for sequestering of Fe and Pb ions from industrial wastewater.

Pore size distributions derived from adsorption isotherms, immersion calorimetry, and isosteric heats: A comparative study

We compare the pore size distribution of a well-characterized activated carbon derived from model-dependent, adsorption integral equation (AIE) methods with those from model-independent, immersion calorimetry and isosteric heat analyses. The AIE approach applied to nitrogen gave a mean pore width of 0.57 nm; the CO2 distribution exhibited wider dispersion. Spherical model application to CO2 and diffusion limitations for nitrogen and argon were proposed as primary reasons for inconsistency. Immersion enthalpy revealed a sharp decrease in available area equivalent to a cut-off due to molecular exclusion when the accessible surface was assessed against probe kinetic diameter. Mean pore width was identified as 0.58 ± 0.02 nm, endorsing the underlying assumptions for the nitrogen-based AIE approach. A comparison of the zero-coverage isosteric heat of adsorption for various non-polar adsorptives by the porous test sample was compared with the same adsorptives in contact with a non-porous reference adsorbent, leading to an energy ratio or adsorption enhancement factor. A linear relationship between the energy ratio and probe kinetic diameter indicated a primary pore size at 0.59 nm. The advantage of this enthalpy, model-independent methods over AIE were due to no assumptions regarding probe molecular shape, and no assumptions for pore shape and/or connectivity.

High-sensitivity differential scanning calorimetry for measurement of steroid entrapment in nebulised liposomes generated from proliposomes

A novel approach to the determination of steroid entrapment in the bilayers of aerosolised liposomes has been introduced using high-sensitivity differential scanning calorimetry (DSC). Proliposomes were dispersed in water within an air-jet nebuliser and the energy produced during atomisation was used to hydrate the proliposomes and generate liposome aerosols. Proliposomes that included the steroid beclometasone dipropionate (BDP) produced lower aerosol and lipid outputs than steroid-free proliposomes. Size analysis and transmission electron microscopy showed an evidence of liposome formation within the nebuliser, which was followed by deaggregation and size reduction of multilamellar liposomes on nebulisation to a two-stage impinger. For each formulation, no difference in thermal transitions was observed between delivered liposomes and those remaining in the nebuliser. However, steroid (5 mole%) lowered the onset temperature and the enthalpy of the pretransition, and produced a similar onset temperature and larger enthalpy of the main transition, with broadened pretransition and main transitions. This indicates that BDP was entrapped and exhibited an interaction with the liposome phospholipid membranes. Since the pretransition was depressed but not completely removed and no phase separation occurred, it is suggested that the bilayers of the multilamellar liposomes can entrap more than 5 mole% BDP. Overall, liposomes were generated from proliposomes and DSC investigations indicated that the steroid was entrapped in the bilayers of aerosolised multilamellar vesicles.

Assessment of resting energy expenditure in pediatric mitochondrial diseases with indirect calorimetry

Mitochondrial diseases (MD) are the most frequent inborn errors of metabolism. In affected tissues, MD can alter cellular oxygen consumption rate leading to potential decreases in whole-body resting energy expenditure (REE), but data on pediatric children are absent. We determined, using indirect calorimetry (IC), whole-body oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory quotient (RQ) and REE in pediatric patients with MD and healthy controls. Another goal was to assess the accuracy of available predictive equations for REE estimation in this patient population. IC data were obtained under fasting and resting conditions in 20 MD patients and 27 age and gender-matched healthy peers. We determined the agreement between REE measured with IC and REE estimated with Schofield weight and FAO/WHO/UNU equations. Mean values of VO2, VCO2 (mL·min-1·kg-1) or RQ did not differ significantly between patients and controls (P = 0.085, P = 0.055 and P = 0.626 respectively). Accordingly, no significant differences (P = 0.086) were found for REE (kcal·day-1 kg-1) either. On the other hand, although we found no significant differences between IC-measured REE and Schofield or FAO/WHO/UNU-estimated REE, Bland-Altman analysis revealed wide limits of agreement and there were some important individual differences between IC and equation-derived REE. VO2, VCO2, RQ and REE are not significantly altered in pediatric patients with MD compared with healthy controls. The energy demands of pediatric patients with MD should be determined based on IC data in order to provide the best possible personalized nutritional management for these children.

Solid Lipid Nanoparticles For Hydrophilic Biotech Drugs: Optimization And Cell Viability Studies (caco-2 & Hepg-2 Cell Lines).

Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

Nitric Oxide-releasing Poly(vinyl Alcohol) Film For Increasing Dermal Vasodilation.

Pathological conditions associated with the impairment of nitric oxide (NO) production in the vasculature, such as Raynaud's syndrome and diabetic angiopathy, have stimulated the development of new biomaterials capable of delivering NO topically. With this purpose, we modified poly(vinyl-alcohol) (PVA) by chemically crosslinking it via esterification with mercaptosuccinic acid. This reaction allowed the casting of sulfhydrylated PVA (PVA-SH) films. Differential scanning calorimetry and X-ray diffractometry showed that the crosslinking reaction completely suppressed the crystallization of PVA, leading to a non-porous film with a homogeneous distribution of -SH groups. The remaining free hydroxyl groups in the PVA-SH network conferred partial hydrophylicity to the material, which was responsible for a swelling degree of ca. 110%. The PVA-SH films were subjected to an S-nitrosation reaction of the -SH groups, yielding a PVA containing S-nitrosothiol groups (PVA-SNO). Amperometric and chemiluminescence measurements showed that the PVA-SNO films were capable of releasing NO spontaneously after immersion in physiological medium. Laser Doppler-flowmetry, used to assess the blood flow in the dermal microcirculation, showed that the topical application of hydrated PVA-SNO films on the health skin led to a dose- and time-dependent increase of more than 5-fold in the dermal baseline blood flow in less than 10min, with a prolonged action of more than 4h during continuous application. These results show that PVA-SNO films might emerge as a new material with potential for the topical treatment of microvascular skin disorders.

Construção de um calorímetro isotérmico diferencial de alta sensibilidade e baixo custo

The high cost of sensitivity commercial calorimeters may represent an obstacle for many calorimetric research groups. This work describes the construction and calibration of a batch differential heat conduction calorimeter with sample cells volumes of about 400 μL. The calorimeter was built using two small high sensibility square Peltier thermoelectric sensors and the total cost was estimated to be about US$ 500. The calorimeter was used to study the excess enthalpy of solution of binary mixtures of liquids, as a function of composition, for the following binary systems of solvents: water + 1,4-dioxane or + dimethylsulfoxide at 298,2 ± 0,5 K.

Efeitos do treinamento com pesos sobre a densidade mineral ossea, força muscular e variaveis funcionais na menopausa

Universidade Estadual de Campinas . Faculdade de Educação Física