Development of magnetically aligned phospholipid bilayers in mixtures of palmitoylstearoylphosphatidylcholine and dihexanoylphosphatidylcholine by solid-state NMR spectroscopy

AbstractThis study reports the solid-state NMR spectroscopic characterization of a long chain phospholipid bilayer system which spontaneously aligns in a static magnetic field. Magnetically aligned phospholipid bilayers or bicelles are model systems which mimic biological membranes for magnetic resonance studies. The oriented membrane system is composed of a mixture of the bilayer forming phospholipid palmitoylstearoylphosphatidylcholine (PSPC) and the short chain phospholipid dihexanoylphosphatidylcholine (DHPC) that breaks up the extended bilayers into bilayered micelles or bicelles that are highly hydrated (approx. 75% aqueous). Traditionally, the shorter 14 carbon chain phospholipid dimyristoylphosphatidylcholine (DMPC) has been utilized as the bilayer forming phospholipid in bicelle studies. Alignment (perpendicular) was observed with a PSPC/DHPC q ratio between 1.6 and 2.0 slightly above Tm at 50°C with 2H and 31P NMR spectroscopy. Paramagnetic lanthanide ions (Yb3+) were added to flip the bilayer discs such that the bilayer normal was parallel with the static magnetic field. The approx. 1.8 (PSPC/DHPC) molar ratio yields a thicker membrane due to the differences in the chain lengths of the DMPC and PSPC phospholipids. The phosphate-to-phosphate thickness of magnetically aligned PSPC/DHPC phospholipid bilayers in the Lα phase may enhance the activity and/or incorporation of different types of integral membrane proteins for solid-state NMR spectroscopic studies

Expression, Purification, and Monitoring of Conformational Changes of hCB2 TMH67H8 in Different Membrane-Mimetic Lipid Mixtures Using Circular Dichroism and NMR Techniques

This work was intended to develop self-assembly lipids for incorporating G-protein coupled receptors (GPCRs) in order to improve the success rate for nuclear magnetic resonance spectroscopy (NMR) structural elucidation. We hereby report the expression and purification of uniformly 15N-labeled human cannabinoid receptor-2 domain in insect cell media. The domain was refolded by screening several membrane mimetic environments. Different q ratios of isotropic bicelles were screened for solubilizing transmembrane helix 6, 7 and 8 (TMH67H8). As the concentration of dimyristoylphosphocholine (DMPC) was increased such that the q ratio was between 0.16 and 0.42, there was less crowding in the cross peaks with increasing q ratio. In bicelles of q = 0.42, the maximum number of cross peaks were obtained and the cross peaks were uniformly dispersed. The receptor domain in bicelles beyond q = 0.42 resulted in peak crowding. These studies demonstrate that GPCRs folding especially in bicelles is protein-specific and requires the right mix of the longer chain and shorter chain lipids to provide the right environment for proper folding. These findings will allow further development of novel membrane mimetics to provide greater diversity of lipid mixtures than those currently being employed for GPCR stability and folding, which are critical for both X-ray and NMR studies of GPCRs

Electrochemical Response of Cells Using Bioactive Plant Isolates

Traditional herbal medical practices continue to be part of the healthcare needs of the world especially residents of sub-Sahara Africa (sSA). However, the mechanism of action of the plant metabolites to elicit their potency continue to be a mystery due to the lack of standardized methods. The mechanism of plant bioactive compounds to cause cell death is gradually being linked to membrane polarization and depolarization behaviour. The current work seeks to probe the electrochemical response of model cells using bioactive compounds captured in bio-zeolites or membrane mimetics. The voltage and current fluctuations emanating from such studies will establish a correlation between cell death and membrane depolarization. It will be a useful biological interface sensing material with the potential to identify plant metabolites that can selectively detect and destroy diseased cells. Several model membranes have already been developed for biomedical applications and this new paradigm will elevate the usefulness of these model systems. The concept was investigated using extracts from Dioclea reflexa (DR) hook which belongs to the leguminous family. There are certain class of compounds in Dioclea reflexa (DR) that have clinical usefulness in both temperate and tropical regions, however the identity of the bioactive compounds responsible for inducing cell death continue to be a major challenge

Capturing Dioclea Reflexa Seed Bioactives on Halloysite Nanotubes and pH Dependent Release of Cargo against Breast (MCF-7) Cancers In Vitro

In this work, optimization parameters were developed to capture plant metabolites from Dioclea Reflexa (DR) seed ex-tracts onto halloysites nanotubes (HNTs). A one-step pool of the crude extracts at neutral pH from the HNT lumen failed to elicit a reduction in breast cancer, Michigan Cancer Foundation-7 (MCF-7) cell viability. However, the pH-dependent elution of metabolites revealed that the acidic pH samples exhibited profound antiproliferative effects on the cancer cells compared to the basic pH metabolites using both trypan blue dye exclusion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability test. pH~5.2 samples demonstrated by half-maximal inhibitory concentration (IC50) of 0.8 mg and a cyclic voltammetry oxidation peak potential and current of 234 mV and 0.45 µA, respectively. This indicates that the cancer cells death could be attributed to membrane polarization/depolarization effects of the sample. Fluorescence-activated cell sorting (FACS) studies confirmed that the plant metabolites affected breast cancer apoptotic signaling pathways of cell death. The studies proved that plant metabolites could be captured using simplified screening procedures for rapid drug discovery purposes. Such procedures, however, would require the integration of affordable analytical tools to test and isolate individual metabolites. Our approach could be an important strategy to create a library and database of bioactive plant metabolites based on pH values

The Influence of Pineapple Leaf Fiber Orientation and Volume Fraction on Methyl Methacrylate-Based Polymer Matrix for Prosthetic Socket Application

This work reports on the use of low-cost pineapple leaf fiber (PALF) as an alternative reinforcing material to the established, commonly used material for prosthetic socket fabrication which is carbon-fiber-reinforced composite (CFRC) due to the high strength and stiffness of carbon fiber. However, the low range of loads exerted on a typical prosthetic socket (PS) in practice suggests that the use of CFRC may not be appropriate because of the high material stiffness which can be detrimental to socket-limb load transfer. Additionally, the high cost of carbon fiber avails opportunities to look for an alternative material as a reinforcement for composite PS development. PALF/Methyl Methacrylate-based (MMA) composites with 0°, 45° and 90° fiber orientations were made with 5–50 v/v fiber volume fractions. The PALF/MMA composites were subjected to a three-point flexural test to determine the effect of fiber volume fraction and fiber orientation on the flexural properties of the composite. The results showed that 40% v/v PALF/MMA composite with 0° fiber orientation recorded the highest flexural strength (50 MPa) and stiffness (1692 MPa). Considering the average load range exerted on PS, the flexural performance of the novel composite characterized in this work could be suitable for socket-limb load transfer for PS fabrication.Applied Science, Faculty ofNon UBCChemical and Biological Engineering, Department ofReviewedFacult

In vitro antibacterial activities of selected TB drugs in the presence of clay minerals against multidrug-resistant strain of Mycobacterium smegmatis

Healing clay is a rich source of diverse minerals. The relevance of these indigenous minerals in the improvement of antibiotic chemotherapy against prevailing bacterial pathogens is yet to be thoroughly explored. In the present study, healing clay from archaeological context was characterized and used in combination with 19 different antibacterial drugs to test their combined in vitro activity against Mycobacterium smegmatis mc2 155 and a multidrug-resistant (MDR) Mycobacterium smegmatis strain. Among the antibiotics tested, the anti-tuberculosis drug, pyrazinamide (Pzd), showed a drastic antimycobacterial activity against Mycobacterium smegmatis mc2 155 in the presence of 5 µg/µL of the healing clay, whereas ribosome targeted inhibitors such as gentamicin showed significant reduction in activity in the presence of the healing clay. The resistance phenotype of the MDR Mycobacterium smegmatis strain to ampicillin and isoniazid was reversed in the presence of the healing clay. The activity of the other antibiotics was either unaffected, enhanced or reduced in the presence of the healing clay. The activity of ampicillin and isoniazid against the MDR strain in the presence of the healing clay suggest that healing clay might be a useful synergy for these antibiotics against MDR Mycobacterium tuberculosis

Mechanical and Structural Characterization of Pineapple Leaf Fiber

Evidence-based research had shown that elevated alkali treatment of pineapple leaf fiber (PALF) compromised the mechanical properties of the fiber. In this work, PALF was subjected to differential alkali concentrations: 1, 3, 6, and 9% wt/wt to study the influence on the mechanical and crystal properties of the fiber. The crystalline and mechanical properties of untreated and alkali-treated PALF samples were investigated by X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FTIR), and tensile testing analysis. The XRD results indicated that crystal properties of the fibers were modified with 6% wt/wt alkali-treated PALF recording the highest crystallinity and crystallite size of 76% and 24 nm, respectively. The FTIR spectra suggested that all alkali-treated PALF samples underwent lignin and hemicellulose removal to varying degrees. An increase in the crystalline properties improved the mechanical properties of the PALF treated with alkali at 6% wt/wt, which has the highest tensile strength (1620 MPa). Although the elevated alkali treatment resulted in decreased mechanical properties of PALF, crystallinity generally increased. The findings revealed that the mechanical properties of PALF not only improve with increasing crystallinity and crystallite size, but are also dependent on the intermediate bond between adjacent cellulose chains.Applied Science, Faculty ofNon UBCChemical and Biological Engineering, Department ofReviewedFacult