Tuning Dunaliella tertiolecta for Enhanced Antioxidant Production by Modification of Culture Conditions.

Microalgae, a popular source of food and bioactive compounds, accumulate antioxidants in response to culture condition stresses. Using a factorial design (3 × 3), the effect of light, temperature, and nitrogen level on chlorophyll and carotenoids, total protein, total phenolic, ascorbate and glutathione content, and enzyme (catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD)) activities in Dunaliella tertiolecta was studied. Data were analysed using Design of Experiments (DoE), and recommendations are made for optimum cultivation conditions to achieve the highest antioxidant content (phenolics, ascorbate and glutathione) or enzyme (CAT, SOD, and POD) activities. This is the first study to apply three levels of three factors during cultivation to tune Dunaliella tertiolecta for optimal antioxidant production.

Current perspectives on supercharging reagents in electrospray ionization mass spectrometry.

In electrospray ionization mass spectrometry (ESI-MS), analytes are introduced into the mass spectrometer in typically aqueous-organic solvent mixtures, including pH modifiers. One mechanism for improving the signal intensity and simultaneously increasing the generation of higher charge-state ions is the inclusion of small amounts (approx. <0.5% v/v mobile phase solution) of charge-inducing or supercharging reagents, such as m-nitrobenzyl alcohol, o-nitrobenzyl alcohol, m-nitrobenzonitrile, m-(trifluoromethyl)-benzyl alcohol and sulfolane. We explore the direct and indirect (colligative properties) that have been proposed as responsible for their modes of action during ESI. Of the many theorized mechanisms of ESI, we re-visit the three most popular and highlight how they are impacted by supercharging observations on small ions to large molecules including proteins. We then provide a comprehensive list of 34 supercharging reagents that have been demonstrated in ESI experiments. We include an additional 19 potential candidate isomers as supercharging reagents and comment on their broad physico-chemical properties. It is becoming increasingly obvious that advances in technology and improved ion source design, analyzers e.g. the use of ion mobility, ion trap, circular dichroism (CD) spectroscopy, together with computer modeling are increasing the knowledge base and, together with the untested isomers and yet-to-be unearthed ones, offer opportunities for further research and application in other areas of polymer research.

Effects of Protein Versus Carbohydrate Supplementation on Markers of Immune Response in Master Triathletes: A Randomized Controlled Trial.

Objective: This study examines the long-term effects of ingesting hydrolyzed beef protein versus carbohydrate on indirect markers of immunity during 10 weeks of endurance training in master-aged triathletes (n = 16, age 35-60 years). Methods: Participants were randomly assigned to either a hydrolyzed beef protein (PRO, n = 8) or nonprotein isoenergetic carbohydrate (CHO, n = 8) condition, which consisted of ingesting 20 g of each supplement, mixed with water, once a day immediately post workout, or before breakfast on nontraining days. Salivary human neutrophil peptides (HNP1-3) were measured before and after performing an incremental endurance test to volitional exhaustion at both pre and post intervention. Additionally, baseline levels of platelets, neutrophils, eosinophil basophils, monocytes, and lymphocytes were determined at pre and post intervention. Results: No significant changes in baseline concentration and secretion rate of salivary HNP1-3 were observed for either treatment. The CHO group showed a nonsignificant decrease in resting HNP1-3 concentrations following the intervention (p = 0.052, effect size d = 0.53). Protein supplementation demonstrated a significant reduction in lymphocyte counts pre to post intervention (mean [SD]: 2.30 [0.57] vs. 1.93 [0.45] 103/mm3, p = 0.046, d = 0.77), along with a moderate but not statistically significant increase (d = 0.75, p = 0.051) of the neutrophil-to-lymphocyte ratio. Conclusions: In master-aged triathletes, postworkout ingestion of only protein, with no carbohydrate, may not be as effective as carbohydrate alone to attenuate negative long-term changes of some salivary and cellular immunological markers. Future studies should consider the co-ingestion of both macronutrients.

Purification of lectin and Kunitz trypsin inhibitor from soya seeds.

The search for potent and selective therapeutic agents is progressing by the study of natural compounds in plants. Plant-derived macromolecules are considered emerging therapeutic agents and an alternative to synthetic and small molecule drugs. Where it has long been known that plants possess medicinal properties, the compounds responsible for their action are in many cases still unknown: often only whole crude plant extracts or fractionated extracts are tested for the ability to inhibit common pathogens. Here, we present a fast protein liquid chromatography method for the separation of crude plant proteins. Kunitz trypsin inhibitor (KTI; 24.2 kDa) and lectin (31 kDa) were purified from Glycine max by liquid extraction followed by ion exchange column chromatography. The need for serial chromatographic separation steps has been eliminated by introducing more complex elution profiles hence reducing cost, time and improving recovery. The identity of KTI-A and lectin was confirmed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-ToF MS). Cell proliferation assays using B16F1 melanoma cells revealed that both KTI and the monomeric lectin retained some antiproliferative activity. This method could be useful for rapid and cost-effective purification of bioactive compounds from plant material.

Effects of Supplementation with Beef or Whey Protein Versus Carbohydrate in Master Triathletes.

OBJECTIVE: The present study compares the effect of ingesting hydrolyzed beef protein, whey protein, and carbohydrate on performance, body composition (via plethysmography), muscular thickness, and blood indices of health, including ferritin concentrations, following a 10-week intervention program. METHODS: After being randomly assigned to one of the following groups-beef, whey, or carbohydrate-24 master-age (35-60 years old) male triathletes (n = 8 per treatment) ingested 20 g of supplement mixed with plain water once a day (immediately after training or before breakfast). All measurements were performed pre- and postinterventions. RESULTS: Only beef significantly reduced body mass (p = 0.021) along with a trend to preserve or increase thigh muscle mass (34.1 ± 6.1 vs 35.5 ± 7.4 mm). Both whey (38.4 ± 3.8 vs 36.9 ± 2.8 mm) and carbohydrate (36.0 ± 4.8 vs 34.1 ± 4.4 mm) interventions demonstrated a significantly (p < 0.05) decreased vastus medialis thickness Additionally, the beef condition produced a significant (p < 0.05) increase in ferritin concentrations (117 ± 78.3 vs 150.5 ± 82.8 ng/mL). No such changes were observed for the whey (149.1 ± 92.1 vs 138.5 ± 77.7 ng/mL) and carbohydrate (149.0 ± 41.3 vs 150.0 ± 48.1 ng/mL) groups. Furthermore, ferritin changes in the beef group were higher than the modification observed in whey (p < 0.001) and carbohydrate (p = 0.025) groups. No differences were found between whey and carbohydrate conditions (p = 0.223). No further changes were observed. CONCLUSION: Ingesting a hydrolyzed beef protein beverage after workout or before breakfast (nontraining days) can be effective in preserving thigh muscle mass and in improving iron status in male master-age triathletes.

Influence of electrolytes and a supercharging reagent on charge state distribution and response of neuropeptide ions generated during positive electrospray ionisation mass spectrometry.

To aid in the detection of trace quantities of neuropeptides in a biological matrix (as saliva), the influence of different electrolytes and a supercharging reagent on the positive electrospray ionisation mass spectrometry (ESI-MS) response was investigated. Ammonium acetate, sodium chloride and sodium bicarbonate (10(-7) M to 10(-3) M) and the supercharging reagent m-nitrobenzyl alcohol (m-NBA) was added to the mobile phase and the effect on the ESI response and charge-states distribution (CSD) was studied in a group of peptides (molecular weight range 2.2kDa to 3.5kDa; CGRP, VIP, GLP1, CRF and PrRP). As expected, the result indicates that the ESI response is affected by the presence of additives: ammonium acetate shifted the observed charge states ratio whereas the addition of m-NBA resulted in the appearance of higher maximum charge state ions. This increase in higher charge state for all the peptide ions, [M + nH]n+ to [M + (n + 1)H](n+1), was atttributed to protonation of the C-terminal. However, when the composition of MeCN in a mobile phase containing m-NBA was increased, an enhancement of the total ion signal was observed for non-polar peptide samples. This is a very interesting observation as this is not observed in samples without m-NBA and could be a result of how these peptide ions are solubilised and positioned relative to the droplet surface/air interface.

Peptide polarity and the position of arginine as sources of selectivity during positive electrospray ionisation mass spectrometry.

Electrospray ionisation (ESI) is a selective process and, for similar sized analytes, the intrinsic properties of the molecules affect the ionisation process and their response. This research sets out to determine the effect of some of these properties in peptides: peptide polarity and the presence of arginine at positions 1 and 4 in the amino acid sequence on the ESI response. Six peptides; molecular mass ranges 1.3-1.6 kDa; substance P (SP) and glutamate fibrinopeptide (GFP) and 3.2-3.7 kDa; calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), glucagon-like peptide 1 (GLP1) and defensin human neutropeptide 2 (DHNP2), were investigated. We have demonstrated that in positive ESI, for similar sized peptides and the same charge state, the responsiveness is in the order: Peptides with N or C terminal arginine > most non-polar peptides > least non-polar peptides. Therefore, arginine at the terminal position is a source of selectivity. Data from matrix-assisted laser desorption ionisation (MALDI) analysis supports that of the ESI experiments: Peptides with a terminal arginine residue generated higher signal intensities. Our observations extend our understanding of the ESI process and provide a rational approach to optimising sensitivity of electrospray conditions where a narrow mass range of peptides are poorly chromatographically resolved. This information will provide for a more effective method development process, especially during label-free quantitative determination of peptides extracted in solution.

The Knoevenagel product of indolin-2-one and ferrocene-1,1'-dicarbaldehyde.

Indolin-2-one (oxindole), (I), undergoes a Knoevenagel condensation with ferrocene-1,1'-dicarbaldehyde, (II), to afford the title complex 3,3'-[(E,E)-ferrocene-1,1'-diyldimethylidyne]diindolin-2-one dichloromethane disolvate, [Fe(C(28)H(20)N(2)O(2))]·2CH(2)Cl(2), (IV). The structure of (IV) contains two ferrocene complex molecules in the asymmetric unit and displays, as expected, intermolecular hydrogen bonding (N-H···O=C) between the indolin-2-one units. Intermolecular π-π stacking interactions are also observed.

Practical considerations in analysing neuropeptides, calcitonin gene-related peptide and vasoactive intestinal peptide, by nano-electrospray ionisation and quadrupole time-of-flight mass spectrometry: monitoring multiple protonations.

We investigated the pre-electrospray ionisation (pre-ESI) factors; analyte concentration (1-2500 ng/mL), concentration of formic acid (FA) in the mobile phase (0.01, 0.1 and 1%), concentration of the organic modifier (acetonitrile 50-90%) and flow rate (<10 µL/min) on the number of multiple protonations and ESI response for two neuropeptides (of ~3.3 kDa molecular mass); calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP). A pH of 3.23 (0.1% FA), nano-flow rate range of 350-750 nL/min and acetonitrile concentration of 50% were optimum for both neuropeptides where the highest intensities were observed. An inverse relationship between decreasing flow rate and ESI response for both peptides was also observed. The quadruply charged ([M+4H](4+)) ion was dominant for CGRP at all analyte concentrations, and also for VIP, but only at the higher analyte concentrations (250-2500 ng/mL); none of the [M+4H](4+), [M+5H](5+) or [M+6H](6+) ions were dominant at the lower concentrations. Linear correlations were obtained for the protonated states and ESI response at analyte concentrations (1-750 ng/mL). Acetonitrile concentration was critical; severe ion suppression was observed for VIP when the concentration of acetonitrile was ≥60%. Ion suppression was also observed for both peptides in an equimolar mixture, with the extent of ion suppression more severe for VIP. Our study concludes that it is important to monitor several protonated species when a single protonated state does not dominate, especially during label-free peptide quantitations.

Multifunctional poly(alkyl methacrylate) films for dental care.

Towards the evaluation of non-permanent dental coatings for their capacity to impart dental-care benefits, thin films of a homologous series of comb-like poly(alkyl methacrylate)s (ethyl to octadecyl) have been deposited, from aqueous latex formulations, onto dentally relevant substrates. AFM studies have shown that the thickness (40-300 nm) and surface roughness (8-12 nm) of coherent polymer films are influenced by the degree of polymerization and by the length of the pendant chain. Of the polymers under consideration, poly(butyl methacrylate) formed a close-packed film that conferred to dental substrates a high degree of inhibition to acid-mediated erosion (about 27%), as evaluated by released-phosphate determinations. The potential utility of the coatings to act as anti-sensitivity barriers has been evaluated by determining the hydraulic conductance of coated bovine-dentine substrates; single treatments of dentine discs with poly(butyl methacrylate) or with poly(ethyl methacrylate) effected mean respective reductions in fluid flow of about 23% with respect to water-treated controls; repeated applications of the poly(butyl methacrylate) latex led to mean reductions in fluid flow of about 80%. Chromometric measurements have shown that pellicle-coated hydroxyapatite discs treated with poly(butyl methacrylate), poly(hexyl methacrylate) or poly(lauryl methacrylate) exhibit significant resistance to staining by food chromogens.

HMGB6 from Arabidopsis thaliana specifies a novel type of plant chromosomal HMGB protein.

The high-mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that act as architectural factors in various nucleoprotein structures, which regulate DNA-dependent processes such as transcription and recombination. Database analyses revealed that in addition to the previously identified HMGB1-HMGB5 proteins, the Arabidopsis genome encodes at least three other family members having the typical overall structure of a central HMG-box DNA binding domain, which is flanked by basic and acidic regions. These novel HMGB proteins display some structural differences, when compared to HMGB1-HMGB5. Therefore, a representative of the identified proteins, now termed HMGB6, was further analyzed. The HMGB6 protein of approximately 27 kDa is the largest plant HMGB protein identified so far. This is essentially due to its unusually extended N-terminal domain of 109 amino acid residues. Subcellular localization experiments demonstrate that it is a nuclear protein. According to CD measurements, HMGB6 has an alpha-helical HMG-box domain. HMGB6 can bind DNA structure-specifically, and it is a substrate for the protein kinase CK2alpha. Because of these features, HMGB6, and presumably its relatives, can be considered members of the plant HMGB protein family. Hence, eight different chromosomal HMGB proteins are expressed in Arabidopsis, and they may serve specialized architectural functions assisting various DNA-dependent processes.