Discrimination of prostate cancer cells and non-malignant cells using secondary ion mass spectrometry

This communication utilises Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) combined with multivariate analysis to obtain spectra from the surfaces of three closely related cell lines allowing their discrimination based upon mass spectral ions

Gating mechanism of elongating β-ketoacyl-ACP synthases.

Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, β-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features in elongating KSs

Comparison on Functional Assays for Gq-Coupled GPCRs by Measuring Inositol Monophospate-1 and Intracellular Calcium in 1536-Well Plate Format

Cell-based functional assays used for compound screening and lead optimization play an important role in drug discovery for G-protein coupled receptors (GPCRs). Cell-based assays can define the role of a compound as an agonist, antagonist or inverse agonist and can provide detailed information about the potency and efficacy of a compound. In addition, cell-based screens can be used to identify allosteric modulators that interact with sites other than the binding site of the endogenous ligand. Intracellular calcium assays which use a fluorescent calcium binding dye (such as Fluo-3, Fluo-4 or Fura-2) have been used in compound screening campaigns to measure the activity of Gq-coupled GPCRs. However, such screening methodologies require a special instrumentation to record the rapid change in intracellular free calcium concentration over time. The radioactive inositol 1,4,5- triphosphate (IP3) assay measures 3H-inositol incorporation and is another traditional assay for the assessment of Gq-coupled GPCR activity, but it is not suitable for screening of large size compound collections because it requires a cell wash step and generates radioactive waste. To avoid these limitations, we have optimized and miniaturized a TR-FRET based IP-One assay that measures inositol monophosphate in a 1536-well plate format. This assay is homogenous, non-radioactive and does not require a kinetic readout. It has been tested with the cell lines expressing M1 acetylcholine, FFAR1, vasopressin V1b, or Neuropeptide S receptors. The activities of antagonists determined in the IP-One assay correlated well with these measured in the intracellular calcium assay while the correlation of agonist activities might vary from cell line to cell line. This IP-One assay offers an alternative method for high throughput screening of Gq-coupled GPCRs without using costly kinetic plate readers

Quantitation of cell surface antigen density by flow cytometry

Correlations between two cellular parameters measured by flow cytometry can be visualized rapidly in contour or isometric data displays. In certain cases, the ratio of two measured parameters has biological meaning and yields the distribution of a new cellular property that cannot be directly measured. In this study, the parameters measured were cell volume and the fluorescence of FITC-labeled antibody to cell surface antigen sites. Cell volume raised to the two-thirds power yields a measure of surface area. On a cell-by-cell basis, the density of a surface antigen is determined by computing the ratio of fluorescence to surface area. Statistical comparisons between calculated density distributions can be made on an absolute as well as a relative basis

Modelling the changes in viscosity during thermal treatment of milk protein concentrate using kinetic data

peer-reviewedThis work aimed to model the effect of heat treatment on viscosity of milk protein concentrate (MPC) using kinetic data. MPC obtained after ultrafiltration was subjected to different time-temperature heat treatment combinations. Heat treatment at high temperature and short time (i.e., 100 or 120 °C×30 s) led to a significant increase in viscosity in MPC systems. Second-order reaction kinetic models proved a better fit than zero- or first-order models when fitted for viscosity response to heat treatment. A distinct deviation in the slope of the Arrhenius plot at 77.9 °C correlated to a significant increase in the rate of viscosity development at temperatures above this, confirming the transition of protein denaturation from the unfolding to the aggregation stage. This study demonstrated that heat-induced viscosity of MPC as a result of protein denaturation/aggregation can be successfully modelled in response to thermal treatment, providing useful new information in predicting the effect of thermal treatment on viscosity of MPC

Effect of pH and heat treatment on viscosity and heat coagulation properties of milk protein concentrate

peer-reviewedThe effect of pH, adjusted using either hydrochloric acid (HCl), citric acid or sodium hydroxide, on calcium ion (Ca2+) activity, and consequent changes in viscosity and heat coagulation time (HCT) of milk protein concentrate (MPC) was investigated. Reducing the pH of MPC dispersions resulted in a reduction in their viscosity, which subsequently increased during heat treatment. The maximum heat stability of MPC was observed at pH 6.7. Reducing the pH of MPC from 6.7 to 6.2 resulted in a significant (P < 0.05) increase in Ca2+ activity, and reduction in HCT. Such changes were more extensive using HCl compared with citric acid. Increasing the pH greater than 6.7 also led to a reduction in HCT but a decrease in Ca2+ activity. These results demonstrate the importance of pH adjustment, and choice of acidulant, on Ca2+ activity, viscosity, and heat coagulation properties of MPC concentrates during processing

Short communication: Multi-component interactions causing solidification during industrial-scale manufacture of pre-crystallized acid whey powders

peer-reviewedAcid whey (AW) is the liquid co-product arising from acid-induced precipitation of casein from skim milk. Further processing of AW is often challenging due to its high mineral content, which can promote aggregation of whey proteins, which contributes to high viscosity of the liquid concentrate during subsequent lactose crystallization and drying steps. This study focuses on mineral precipitation, protein aggregation, and lactose crystallization in liquid AW concentrates (∼55% total solids), and on the microstructure of the final powders from 2 independent industrial-scale trials. These AW concentrates were observed to solidify either during processing or during storage (24 h) of pre-crystallized concentrate. The more rapid solidification in the former was associated with a greater extent of lactose crystallization and a higher ash-to-protein ratio in that concentrate. Confocal laser scanning microscopy analysis indicated the presence of a loose network of protein aggregates (≤10 µm) and lactose crystals (100–300 µm) distributed throughout the solidified AW concentrate. Mineral-based precipitate was also evident, using scanning electron microscopy, at the surface of AW powder particles, indicating the formation of insoluble calcium phosphate during processing. These results provide new information on the composition- and process-dependent physicochemical changes that are useful in designing and optimizing processes for AW

Influence of protein standardisation media and heat treatment on viscosity and related physicochemical properties of skim milk concentrate

peer-reviewedThe effects of heat treatment and protein standardisation on the physical properties of skim milk concentrates were determined. Protein standardisation was carried out by the addition of lactose or milk permeate to skim milk. Unstandardised and standardised skim milk was subjected to heat treatment temperatures of 90 or 120 °C prior to evaporation whereafter the solids content was increased to 46% (w/w). Viscosity data showed non-standardised concentrates had the highest viscosity, followed by skim standardised with milk permeate followed by that standardised with lactose. Thermal treatment at 120 °C also resulted in a higher viscosity than that at 90 °C for all concentrates. Particle size data of evaporated skim milk showed a bimodal size distribution for skim milk standardised with liquid milk permeate, compared with monomodal distribution profiles for unstandardised skim milk and lactose standardised skim milk. Overall, this study showed that protein standardisation and standardisation media significantly affected concentrate properties

NASA Light Emitting Diode Medical Applications from Deep Space to Deep Sea

This work is supported and managed through the NASA Marshall Space Flight Center-SBIR Program. LED-technology developed for NASA plant growth experiments in space shows promise for delivering light deep into tissues of the body to promote wound healing and human tissue growth. We present the results of LED-treatment of cells grown in culture and the effects of LEDs on patients’ chronic and acute wounds. LED-technology is also biologically optimal for photodynamic therapy of cancer and we discuss our successes using LEDs in conjunction with light-activated chemotherapeutic drugs