Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion

This is a comprehensive study of protein-mediated membrane fusion through single-molecule fluorescence resonance energy transfer (smFRET). Membrane fusion is one of the important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. For example, exocytosis, fertilization of an egg by a sperm and communication between neurons are a few among many processes that rely on some form of fusion. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) play a central role in fusion processes which is also regulated by many accessory proteins, such as synaptotagmin, complexin and Munc18. By a new lipid mixing method at the single-vesicle level, we are able to accurately detect different stages of SNARE-mediated membrane fusion including docking, hemi and full fusion via FRET value of single donor/acceptor vesicle pair. Through this single-vesicle lipid mixing assay, we discovered the vesicle aggregation induced by C2AB/Ca2+, the dual function of complexin, and the fusion promotion role of Munc18/SNARE-core binding mode. While this new method provides the information regarding the extent of the ensemble lipid mixing, the fusion pore opening between two vesicular cavities and the interaction between proteins cannot be detected. In order to overcome these limitations, we then developed a single-vesicle content mixing method to reveal the key factor of pore expansion by detecting the FRET change of dual-labeled DNA probes encapsulated in vesicles. Through our single-vesicle content mixing assay, we found the fusion pore expansion role of yeast SNAREs as well as neuronal SNAREs plus synaptotagmin 1.

Effects of various salt purity levels on lipid oxidation and sensory characteristics of ground turkey and pork

Salt use in meat products is changing. Consumers desire sea salt which may also contain trace metals and the government is demanding a reduction in sodium. Therefore a need exists to understand how varying impurity levels in salt affect meat quality. This study evaluated the effects of various salt preparations on lipid oxidation, sensory characteristics, protein extractability, and bind strength of ground turkey and pork. This study was a completely randomized design with 5 treatment groups and 6 replications in 2 species. Ground, turkey and pork meat was formulated into one hundred and fifty gram patties with sodium chloride (1%) containing varying amounts of metal impurities (copper, iron, and manganese). Samples were randomly assigned to frozen storage periods of 0, 3, 6, and 9 weeks. After storage, samples were packaged in PVC overwrap and stored under retail display for 5 days. Samples were evaluated for proximate analysis to ensure the fat content was similar for all of the starting material.Thiobarbituric acid reactive substances (TBARS) were determined on raw and cooked samples to evaluate lipid oxidation. A trained six member sensory panel evaluated the samples at each storage period for saltiness, off flavor, and oxidized odor. Break strength was conducted using a Texture Analyzer and compared with salt soluble proteins (increasing salt concentrations) to evaluate protein extractability characteristics. Statistical analyses were conducted using the MIXED procedure of SAS within repeated measures over time where appropriate. No significant differences were observed among the salt treatments for raw and cooked TBARS when the control group was removed (P>0.05). Sensory panelists detected increased levels of off flavor and oxidized odor over the entire storage duration. Less force was required to break the patties from the control group when compared with the salt treatments (P<0.05). As salt concentration increased salt-soluble protein extraction increased, but there was no effect of salt type. Overall, no meaningful statistical differences among the various salt treatments were observed for all of the parameters evaluated for turkey and pork. Salt at a 1% inclusion rate containing varying levels of copper, iron, and manganese impurities in ground turkey thigh meat and ground pork served as a prooxidant. However, if a meat processor uses a 1% inclusion rate of salt in turkey and pork regardless of impurities included, it is unlikely that differences in shelf life or protein functionality would be observed.