RAS-RAF-miR-296-3p signaling axis increases Rad18 expression to augment radioresistance in pancreatic and thyroid cancers.

Oncogenic RAS and RAF signaling has been implicated in contributing to radioresistance in pancreatic and thyroid cancers. In this study, we sought to better clarify molecular mechanisms contributing to this effect. We discovered that miRNA 296-3p (miR-296-3p) is significantly correlated with radiosensitivity in a panel of pancreatic cancer cells, and miR-296-3p is highly expressed in normal cells, but low in cancer cell lines. Elevated expression of miR-296-3p increases radiosensitization while decreasing the expression of the DNA repair enzyme RAD18 in both pancreatic and thyroid cancer cells. RAD18 is overexpressed in both pancreatic and thyroid tumors compared to matched normal controls, and high expression of RAD18 in tumors is associated with poor prognostic features. Modulating the expression of mutant KRAS in pancreatic cancer cells or mutant BRAF in thyroid cancer cells demonstrates a tight regulation of RAD18 expression in both cancer types. Depletion of RAD18 results in DNA damage and radiation-induced cell death. Importantly, RAD18 depletion in combination with radiotherapy results in marked and sustained tumor regression in KRAS mutant pancreatic cancer orthotopic tumors and BRAF mutant thyroid heterotopic tumors. Overall, our findings identify a novel coordinated RAS/RAF-miR-296-3p-RAD18 signaling network in pancreatic and thyroid cancer cells, which leads to enhanced radioresistance.

Tuning Catalyst Selectivity for Ammonia vs Hydrogen: An Investigation into the Coprecipitation of Mo and Fe Sulfides.

Iron sulfides are key materials in metalloprotein catalysis. One interesting aspect of iron sulfides in biology is the incorporation of secondary metals, for example, Mo, in nitrogenase. These secondary metals may provide vital clues as to how these enzymes first emerged in nature. In this work, we examined the materials resulting from the coprecipitation of molybdenum with iron sulfides using X-ray absorption spectroscopy (XAS). The materials were tested as catalysts, and direct reductants using nitrite (NO2-) and protons (H+) as test substrates. It was found that Mo will coprecipitate with iron as sulfides, however, in distinct ways depending on the stoichiometric ratios of Mo, Fe, and HS-. It was observed that the selectivity of reduction products depends on the amount of molybdenum, with the presence of approximately at 10% Mo optimizing ammonium/ammonia (NH4+/NH3) production from NO2- and minimizing competitive hydrogen (H2) formation from protons (H+) with a secondary reductant.

Antibody Production Remains Intact Despite Loss of Bone Marrow B cells in Murine Norovirus Infected Stat1-/- Mice.

Murine norovirus (MNV), which can be used as a model system to study human noroviruses, can infect macrophages/ monocytes, neutrophils, dendritic, intestinal epithelial, T and B cells, and is highly prevalent in laboratory mice. We previously showed that MNV infection significantly reduces bone marrow B cell populations in a Stat1-dependent manner. We show here that while MNV-infected Stat1-/- mice have significant losses of bone marrow B cells, splenic B cells capable of mounting an antibody response to novel antigens retain the ability to expand. We also investigated whether increased granulopoiesis after MNV infection was causing B cell loss. We found that administration of anti-G-CSF antibody inhibits the pronounced bone marrow granulopoiesis induced by MNV infection of Stat1-/- mice, but this inhibition did not rescue bone marrow B cell losses. Therefore, MNV-infected Stat1-/- mice can still mount a robust humoral immune response despite decreased bone marrow B cells. This suggests that further investigation will be needed to identify other indirect factors or mechanisms that are responsible for the bone marrow B cell losses seen after MNV infection. In addition, this work contributes to our understanding of the potential physiologic effects of Stat1-related disruptions in research mouse colonies that may be endemically infected with MNV.

Conductive, Acid-Doped Polyaniline Electrospun Nanofiber Gas Sensing Substrates Made Using a Facile Dissolution Method.

A novel dissolution method that allows for the total solvation of high-concentration, high-molecular-weight polyaniline (PANi) doped with (+)-camphor-10-sulfonic acid (CSA) is reported. Preparation of 12-16 wt % 65,000 Da PANi solutions in N,N-dimethylformamide is achievable using a simple one-pot method. Doped polyaniline solutions in common organic solvents were processed into nanofibers using a convenient single-nozzle electrospinning technique. The electrospinning of PANi-CSA into nanofibrous membranes generated substrates that were subsequently employed in colorimetric gas sensing. These substrates demonstrated linearity of response upon exposure to 50-5500 ppm ammonia at ambient (50 ± 10% RH) and high (80% RH) humidity.

The role of lecithin degradation on the pH dependent stability of halofantrine encapsulated fat nano-emulsions.

We report on the successful incorporation of the antimalarial drug, halofantrine, into laboratory based soybean oil emulsions which were designed to mimic the commercially available parenteral fat emulsion, Intralipid®. A high pH (minimum of pH 9, preferable pH of 11) was required for the drug laden emulsion to remain stable on storage and also to resist breaking under various stresses. Ageing of lecithin samples on storage was noted to result in degradation and a decrease in pH. We argue that this is the main reason for a similar decrease in pH for lecithin based emulsions and subsequent instability in drug laden emulsions. As expected, incorporation of the drug (halofantrine) resulted in lower stability. The (intensity weighted) particle size increased from 281nm for the drug free emulsion to 550nm following a loading of 1gL-1 of halofantrine, indicative of a lowering in stability and this was reflected in a shorter shelf life. Interestingly, incorporation of even higher concentrations of drug then resulted in better stability albeit never as stable as the drug free emulsion. We also report on unusual and complex surface tension behaviour for fresh lecithin where multiple critical concentration points were observed.

Microwave-assisted microemulsion technique for production of miconazole nitrate- and econazole nitrate-loaded solid lipid nanoparticles.

The microwave-assisted production of solid lipid nanoparticles (SLNs) is a novel technique reported recently by our group. The small particle size, solid nature and use of physiologically well-tolerated lipid materials make SLNs an interesting and potentially efficacious drug carrier. The main purpose of this research work was to investigate the suitability of microwave-assisted microemulsion technique to encapsulate selected ionic drug substances such as miconazole nitrate and econazole nitrate. The microwave-produced SLNs had a small size (250-300nm), low polydispersity (<0.20), high encapsulation efficiency (72-87%) and loading capacity (3.6-4.3%). Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) studies suggested reduced crystallinity of stearic acid in SLNs. The release studies demonstrated a slow, sustained but incomplete release of drugs (<60% after 24h) from microwave-produced SLNs. Data fitting of drug release data revealed that the release of both drugs from microwave-produced SLNs was governed by non-Fickian diffusion indicating that drug release was both diffusion- and dissolution- controlled. Anti-fungal efficacy of drug-loaded SLNs was evaluated on C. albicans. The cell viability studies showed that cytotoxicity of SLNs was concentration-dependent. These encouraging results suggest that the microwave-assisted procedure is suitable for encapsulation of ionic drugs and that microwave-produced SLNs can act as potential carriers of antifungal drugs.

Microwave-assisted formulation of solid lipid nanoparticles loaded with non-steroidal anti-inflammatory drugs.

Stearic acid-based solid lipid nanoparticles (SLNs) were prepared using the microwave assisted one-pot microemulsions procedure pioneered by our group. In this study, non-steroidal anti-inflammatory drugs (NSAIDs) including indomethacin, ketoprofen and nimesulide were selected as ideal "test" drugs, based on their poor water solubility. The model drugs were incorporated within the SLNs by the microwave-assisted procedure at the time of SLN production. The microwave-produced drug-loaded SLNs were evaluated in terms of their physicochemical characteristics, drug release behavior and their uptake into against A549 cell line (human lung epithelial cells). The microwave-produced drug-loaded SLNs had a small particle size distribution, negative zeta potential and high encapsulation efficiency. The drug release studies were consistent with a core-shell structure of SLNs (probably a drug-loaded shell) which results in biphasic drug release from the SLNs. The drug release kinetics suggested a good fit of the release data to the Makoid-Banakar model and was governed by Fickian diffusion. The drug-loaded SLNs showed concentration-dependent cytotoxicity and reduced IL-6 and IL-8 secretion in lipopolysaccharide-induced cells. All of the above findings suggest that the microwave-produced SLNs could be promising drug carriers of NSAIDs and will further facilitate their development for topical, oral and/or nasal administration.

Transport of stearic acid-based solid lipid nanoparticles (SLNs) into human epithelial cells.

Development of drug delivery systems, as much as the drug molecule itself, is an important consideration for improving drug absorption and bioavailability. The mechanisms by which drug carriers enter target cells can differ depending on their size, surface properties and components. Solid lipid nanoparticles (SLNs) have gained an increased attention in recent years and are the drug carriers of interest in this paper. They are known to breach the cell-membrane barrier and have been actively sought to transport biomolecules. Previous studies by our group, and also other groups, provided an extensive characterization of SLNs. However, few studies have investigated the uptake of SLNs and these have had limited mechanistic focus. The aim of this work was to investigate the pathway of uptake of SLNs by human epithelial cells i.e., lung A549 and cervical HeLa cells. To the best of our knowledge, this is first study that investigates the cellular uptake of SLNs by human epithelial cells. The mechanism of cellular uptake was deciphered using pharmacologic inhibitors (sucrose, potassium-free buffer, filipin and cytochalasin B). Imaging techniques and flow assisted cell sorting (FACS) were used to assess the cellular uptake of SLNs loaded with rhodamine 123 as a fluorescent probe. This study provided evidence that the cellular uptake of SLNs was energy-dependent, and the endocytosis of SLNs was mainly dependent on clathrin-mediated mechanisms. The establishment of entry mechanism of SLNs is of fundamental importance for future facilitation of SLNs as biological or drug carriers.

The role of metal ion-ligand interactions during divalent metal ion adsorption.

A suite of seven different divalent metal ions (Ca(II), Cd(II), Cu(II), Mg(II), Ni(II), Pb(II), Zn(II)) was adsorbed from solution onto two Fe2O3 samples, quartz SiO2 and three different amphoteric polystyrene latices (containing amine and carboxyl functional groups). For the metal oxides, a high correlation was observed between the pH at which 50% of the metal was removed from solution (pH50) and the first hydrolysis constant for the metal ion (pK1). For the polystyrene latices, a much higher correlation was observed between the pH50 and pKc (equilibrium constant describing metal-carboxyl affinity) as opposed to pK1. These observations provide evidence of a strong relationship that exists between a metal's affinity for a particular ligand in solution and for that metal ion's affinity for the same ligand present as part of an adsorbing surface. The isoelectric point of the amphoteric latex surface can be increased by decreasing the carboxyl content of the latex surface. For all 7 metal ions, this resulted in a substantial decrease, for any given pH, in adsorption. We suggest that this may be partly due to the decreased carboxyl content, but is dominantly attributable to the presence of less favorable electrostatic conditions. This, in turn, demonstrates that electrostatics play a controlling role in metal ion adsorption onto amphoteric latex surfaces and, in addition to the nature of the metal ion, also controls the pH at which adsorption takes place.

Physicochemical characterization of solid lipid nanoparticles (SLNs) prepared by a novel microemulsion technique.

HYPOTHESIS: Solid lipid nanoparticles (SLNs) produced by conventional microemulsion techniques using thermal heat have specific limitations (e.g. high polydispersity, instability and low encapsulation). Replacing thermal heat with microwave heat may produce SLNs which overcome some of these limitations. EXPERIMENTS: Stearic acid-based SLNs prepared with Tween® 20 as the emulsifier were chosen as the optimum formulation to encapsulate and potentially deliver the antibacterial drug tetracycline. All formulations were characterized for their particle size, zeta potential, encapsulation efficiency, loading capacity, thermal and X-ray diffraction analyses. Short-term stability and in vitro drug studies were also performed. FINDINGS: Microwave heating helps to overcome several disadvantages associated with thermal heating (nonuniform, inefficient and slow) and results in improved particle characteristics. There is thus the potential for new opportunities in the development of colloidal carriers. The particle sizes of microwave-produced SLNs were in the desired nanometer range (200-250 nm) with both lower size and lower polydispersity than the conventional SLNs. We take this as an indication of improved stability; however zeta potential measurements were not different, indicating similar stability. True stability testing (visual observation with time) did show that the microwave-induced SLNs were found to be more stable, particularly when refrigerated. The microwave-produced SLNs also demonstrated improved encapsulation efficiency and loading capacity. Thermal and diffraction analysis confirmed a lowered crystallinity of stearic acid with successful incorporation of tetracycline into the SLNs. In vitro release studies indicated that, after an initial burst release, SLNs could provide prolonged release of tetracycline. The presence of tetracycline and non-toxicity of carriers towards microbes was confirmed by antimicrobial susceptibility tests.