Validation of a Simple HPLC-Based Method for Lysine Quantification for Ruminant Nutrition

Robust and selective quantification methods are required to better analyze feed supplementation effectiveness with specific amino acids. In this work, a reversed-phase high-performance liquid chromatography method with fluorescence detection is proposed and validated for lysine quantification, one of the most limiting amino acids in ruminant nutrition and essential towards milk production. To assess and widen method applicability, different matrices were considered: namely Li₂CO₃ buffer (the chosen standard reaction buffer), phosphate buffer solution (to mimic media in cellular studies), and rumen inoculum. The method was validated for all three matrices and found to be selective, accurate (92% ± 2%), and precise at both the inter- and intra-day levels in concentrations up to 225 µM, with detection and quantification limits lower than 1.24 and 4.14 µM, respectively. Sample stability was evaluated when stored at room temperature, 4 °C, and −20 °C, showing consistency for up to 48 h regardless of the matrix. Finally, the developed method was applied in the quantification of lysine on real samples. The results presented indicate that the proposed method can be applied towards free lysine quantification in ruminant feeding studies and potentially be of great benefit to dairy cow nutrition supplementation and optimization.     

Nonpolar, polar, and associating solutes in ionic liquids

The existence of microphase segregation between polar and nonpolar domains in ionic liquids changes the way in which solvation can be understood in these media. Here, we perform a structural analysis on the solvation of nonpolar, polar, and associating solutes in imidazolium-based ionic liquids, where this novel way of understanding their nature as microsegregated solvents is correlated with their ability to interact with different species in diverse and complex ways.     

Kinetics independent spectrometric analysis using non-linear calibration modelling and exploitation of concentration gradients generated by a flow-batch system for albumin and total protein determination in blood serum

An automatic method for kinetics independent spectrometric analysis is proposed in this study. It uses a non-linear calibration model that explores concentration gradients generated by a flow-batch analyser (FBA) for the samples, dye, and the single standard solution. The procedure for obtaining the gradients of the dye and standard solution is performed once at the beginning of analysis. The same procedure is applied thereafter for each sample. For illustration, the proposed automatic methodology was applied to determine total protein and albumin in blood serum by using the Biuret and Bromocresol Green (BCG) methods. The measurements were made by using a laboratory-made photometer based on a red and green bicolour LED (Light-Emitting Diode) and a phototransistor, coupled to a “Z” form flow cell. The sample throughput was about 50 h⁻¹ for albumin and 60 h⁻¹ for total protein, consuming about 7 μL of sample, 2.6 mL of BCG and 1.2 mL of biuret reagents for each determination. Applying the paired t-test for results from the proposed analyser and the reference method, no statistic differences at 95% confidence level were found. The absolute standard deviation was usually smaller than 0.2 g dL⁻¹. The proposed method is valuable for the determination of total protein and albumin; and can also be used in other determinations where kinetic effects may or may not exist.     

'Model your genes the mathematical way'--a mathematical biology workshop for secondary school teachers

This article describes a mathematical biology workshop givento secondary school teachers of the Danville area in Virginia,USA. The goal of the workshop was to enable teams of teacherswith biology and mathematics expertise to incorporate lessonplans in mathematical modelling into the curriculum. The biologicalfocus of the activities is the lactose operon in Escherichiacoli, one of the first known intracellular regulatory networks.The modelling approach utilizes Boolean networks and tools fromdiscrete mathematics for model simulation and analysis. Theworkshop structure simulated the team science approach commonin today's practice in computational molecular biology and thusrepresents a social case study in collaborative research. Theworkshop provided all the necessary background in molecularbiology and discrete mathematics required to complete the project.The activities developed in the workshop show students the valueof mathematical modelling in understanding biochemical networkmechanisms and dynamics. The use of Boolean networks, ratherthan the more common systems of differential equations, makesthe material accessible to students with a minimal mathematicalbackground. High school students can be exposed to the excitement of mathematicalbiology from both the biological and mathematical point of view.Through the development of instructional modules, high schoolbiology and mathematics courses can be joined without havingto restructure the curriculum for either subject. The relevanceof an early introduction to mathematical biology allows studentsnot only to learn curriculum material in a innovative setting,but also creates an awareness of new educational and careeropportunities that are arising from the interconnections betweenbiological and mathematical sciences. The materials used in this workshop are available at a websitecreated by the directors: http://polymath.vbi.vt.edu/mathbio2006/.     

Thermodynamic characterization of the interaction behavior of a hydrophobically modified polyelectrolyte and oppositely charged surfactants in aqueous solution: effect of surfactant alkyl chain length

We have used a precision isothermal titration microcalorimeter (ITC) to measure the enthalpy curves for the interaction of a hydrophobically modified polyelectrolyte (D40OCT30) with oppositely charged surfactants (SC(n)S) in aqueous solution. D40OCT30 is a newly synthesized polymer based on dextran having pendant N-(2-hydroxypropyl)-N,N-dimethyl-N-octylammonium chloride groups randomly distributed along the polymer backbone with degree of substitution of 28.1%. The employed anionic surfactants are sodium octyl sulfate (SC(8)S) and sodium tetradecyl sulfate (SC(14)S). Microcalorimetric results along with turbidity and kinematic viscosity measurements demonstrate systematically the thermodynamic characterization of the interaction of D40OCT30/SC(n)S. A three-dimensional diagram with the derived phase boundaries is drawn to describe the effect of the alkyl chain length of surfactant and of the ratio between surfactant and pendant groups on the interaction. A more complete picture of the interaction mechanism for D40OCT30/SC(n)S systems is proposed here.     

Structures and vibrational frequencies of vanadium (V) oligomers: An ab initio study using effective core potentials

Ab initio molecular geometries and vibrational frequencies of various isolated vanadate species (VO³⁻₄, HVO²⁻₄, H₂VO⁻₄, and V₂O⁴⁻₇) were calculated using different pseudopotentials. The relative merits of these were assessed by comparing the calculated molecular parameters with the corresponding values obtained from calculations at all-electron levels and, whenever available, from X-ray studies for the salts. The calculations were extended to higher oligomers (V₃O⁵⁻₁₀, V₄O⁶⁻₁₃, and V₄O⁴⁻₁₂) using the pseudopotential whose basis functions are (10s5p5d)/[2s1p1d] (55/5/5) on vanadium and (4s4p)/[2s2p] (31/31) on oxygen, which yielded the best compromise between accuracy and computational effort. The results indicate a linear centrosymmetric geometry for the isolated V₂O⁴⁻₇ anion. The higher oligomers have less than 180° V(SINGLE BOND)O(SINGLE BOND)V angles, except the noncyclic tetraoligomer which has a linear central V(SINGLE BOND)O(SINGLE BOND)V angle (180°). The cyclic V₄O⁴⁻₁₂ species presents a planar structure with all the vanadium and bridging oxygen atoms in the same plane. This structure was alrea dy reported for the [(CH₃)CNH₃][V₄O₁₂] salt. The results suggest a lower stability of the linear V₄O⁶⁻₁₃ species, in agreement with previous reports. © 1996 by John Wiley & Sons, Inc.     

The ionic strength effect on microcystin and natural organic matter surrogate adsorption onto PAC

This work aims to contribute to a better understanding of the ionic strength effect on microcystin and natural organic matter (NOM) surrogate adsorption by analyzing the importance of adsorbate molecular size, and surface concentration. Adsorption kinetics and/or isotherms were performed on PAC Norit SA-UF for four microcystin variants (MC-LR, MC-LY, MC-LW, MC-LF), and three NOM surrogates (salicylic acid (SA), tannic acid (TA), Aldrich humic acid (AHA)) at different solution ionic strengths. Results showed that the ionic strength effect depends upon the adsorbate surface concentration, cation charge (mono or divalent), and adsorbate molecular size. Potassium seemed not to affect the MC-LR adsorption, while calcium enhanced MC-LR kinetics and adsorption capacity. K+ and, particularly, Ca2+ improved the adsorption kinetics of the other microcystin variants. For identical surface concentration and ionic strength, the impact of K+ and Ca2+ on NOM surrogates depended on the adsorbate molecular size: K+ effect was only observed for AHA, whereas Ca2+ caused no effect on SA adsorption, slightly enhanced TA adsorption, and greatly enhanced AHA adsorption. MC-LR isotherms with two salt concentrations (KCl or CaCl2) indicated that, for the studied range of equilibrium surface concentration (5.3-18.7 mg/g), an enhanced adsorption regime prevails, and no transition regime was observed.     

The choroid plexus response to a repeated peripheral inflammatory stimulus

Background  

Chronic systemic inflammation triggers alterations in the central nervous system that may relate to the underlying inflammatory component reported in neurodegenerative disorders such as multiple sclerosis and Alzheimer's disease. However, it is far from being understood whether and how peripheral inflammation contributes to induce brain inflammatory response in such illnesses. As part of the barriers that separate the blood from the brain, the choroid plexus conveys inflammatory immune signals into the brain, largely through alterations in the composition of the cerebrospinal fluid.     

Synthesis and NMR structure of p41icf,a potent inhibitor of human cathepsin L

The total synthesis and structural characterization of the MHCII-associated p41 invariant chain fragment (P41icf) is described. P41icf plays a crucial role in the maturation of MHC class II molecules and antigen processing, acting as a highly selective cathepsin L inhibitor. P41icf synthesis was achieved using a combined solid-phase/solution approach. The entire molecule (65 residues, 7246 Da unprotected) was assembled in solution from fully protected peptides in the size range of 10 residues. After deprotection, oxidative folding in carefully adjusted experimental conditions led to the completely folded and functional P41icf with a disulfide pairing identical to that of native P41icf. CD, NMR, and surface plasmon resonance (SPR) were used for the structural and functional characterization of synthetic P41icf. CD thermal denaturation showed clear cooperative behavior. Tight cathepsin L binding was demonstrated by SPR. (1)H NMR spectroscopy at 800 MHz of unlabeled P41icf was used to solve the three-dimensional structure of the molecule. P41icf behaves as a well-folded protein domain with a topology very close to the crystallographic cathepsin L-bound form.     

Synthesis of chiral 2-(4-phenoxyphenylsulfonylmethyl)thiiranes as selective gelatinase inhibitors

[reactions: see text] Compound 1, 2-(4-phenoxyphenylsulfonylmethyl)thiirane, is a selective inhibitor of gelatinases, which is showing high promise in studies of animal models for cancer metastasis and stroke. The (R)-1 and (S)-1 enantiomers of compound 1 were each synthesized in this study and were shown to be equally active in inhibition of gelatinases.