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A need exists for robust and cost-effective assays to detect a single or small set of actionable point mutations, or a complete set of clinically informative mutant alleles. Knowledge of these mutations can be used to alert the clinician to a rare mutation that might necessitate more aggressive clinical monitoring or a personalized course of treatment. An example is BRAF, a (proto)oncogene susceptible to either common or rare mutations in codon V600 and adjacent codons. We report a diagnostic technology that leverages the unique capabilities of droplet digital PCR to achieve not only accurate and sensitive detection of BRAF(V600E) but also all known somatic point mutations within the BRAF V600 codon. The simple and inexpensive two-well droplet digital PCR assay uses a chimeric locked nucleic acid/DNA probe against wild-type BRAF and a novel wild-type-negative screening paradigm. The assay shows complete diagnostic accuracy when applied to formalin-fixed, paraffin-embedded tumor specimens from metastatic colorectal cancer patients deficient for Mut L homologue-1.

Surface induced thrombus generation is a major clinical concern associated with vascular medical devices and implants. Here, we show that high graft density hydrophilic non-charged poly (N,N-dimethylacrylamide) (PDMA) brushes prevent the initiation of blood coagulation on synthetic surfaces. Using a multi-faceted analysis approach, we have identified that PDMA brushes greater than 0.27 chains/nm(2) graft density showed this highly desired property. Non-specific protein adsorption is greatly reduced on high density brushes compared to bare surface as evident from isothermal titration calorimetry, gel electrophoresis, and proteomic analyses. We have identified approximately 129 proteins of various types on bare and PDMA brush coated surfaces at a range of surface concentrations. Thromboelastography, platelet activation, and aggregation analyses show that only high graft density brushes are neutral to blood coagulation. Since the polymer brush synthesis can be adapted to most currently used biomedical materials, these results have significant impact in the design of highly hemocompatible surfaces.

Summary Assembly of the type-III secretion apparatus, which translocates proteins through both membranes of Gram-negative bacterial pathogens into host cells, requires the formation of an integral outer-membrane secretin ring. Typically, a small lipidated pilot protein is necessary for the stabilization and localization of this ring. Using NMR spectroscopy, we demonstrate that the C-terminal residues 553–570 of the Shigella flexneri secretin MxiD encompass the minimal binding domain for its cognate pilot MxiM. Although unstructured in isolation, upon complex formation with MxiM, these residues fold into an amphipathic turn-helix motif that caps the elongated hydrophobic cavity of the cracked β-barrel pilot. Along with a rearrangement of core aromatic residues, this prevents the binding of lipids within the cavity. The mutually exclusive association of lipids and MxiD with MxiM establishes a framework for understanding the role of a pilot in the outer-membrane insertion and multimerization of the secretin ring.

A model of chiral ligand-exchange chromatography (CLEC) is presented that combines the non-ideal equilibrium-dispersion equation for solute transport with equations describing all chemical equilibria within the column. The model connects elution band profiles to the time and space resolved formation of diastereomeric complexes in both the mobile and stationary phases, thereby providing insights into the overall separation mechanism. The stoichiometries and formation constants for all equilibrium complexes formed in the mobile phase are taken from standard thermodynamic databases and independent potentiometric titration experiments. Formation constants for complexes formed with the stationary phase ligand are determined from potentiometric titration data for a water-soluble analogue of the ligand. Together this set of pure thermodynamic parameters can be used to calculate the equilibrium composition of the system at any operating condition. The model includes a temperature-dependent pure-component parameter, determined by regression to a single elution band for the pure component, that corrects for subtle effects associated with immobilizing the ligand (i.e., the chiral selector) onto the stationary phase. Model performance is assessed through comparison with chromatograms for two hydrophobic amino acid racemates loaded on the Nucleosil Chiral-1 CLEC column. The model is also applied to a restricted optimization of column operating conditions to assess its predictive power. In both cases, model predictions compare well with experiment while also providing a molecular understanding of the separation process and its dependence on column operating conditions.

A novel inexpensive affinity purification technology is described based on recombinant expression in Escherichia coli of the polypeptide or protein target fused through its N-terminus to TmXyn10ACBM9-2 (CBM9), the C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima . Measured association constants ( K a ) for adsorption of CBM9 to insoluble allomorphs of cellulose are between 2×10 5 and 8×10 6 M −1 . CBM9 also binds a range of soluble sugars, including glucose. As a result, a 1 M glucose solution is effective in eluting CBM9 and CBM9-tagged fusion proteins from a very inexpensive commercially-available cellulose-based capture column. A processing site is encoded at the C-terminus of the tag to facilitate its rapid and quantitative removal by Factor X a to recover the desired target protein sequence following affinity purification. Fusion of the CBM9 affinity tag to the N-terminus of green fluorescent protein (GFP) from the jellyfish, Aquorin victoria , is shown to yield >200 mg l −1 of expressed soluble fusion protein that can be affinity separated from clarified cell lysate to a purity of >95% at a yield of 86%.

Ligand-exchange chiral extraction (LEXCEX) is an emerging technology for large-scale continuous resolution of enantiomers of amino acids and a wide range of chiral therapeutics and drug precursors. LEXCEX is based on the ability of a chiral ligand (Li), solubilized in the non-aqueous phase of a water/alcohol two-phase system through complexation with a transition metal ion (i.e., Cu), to preferentially extract one enantiomer (En) into the organic phase through formation of a ternary electroneutral complex. Here we show that the efficiency of the extraction depends, often strongly, on a number of process variables, including the selectivity of the ligand, the solubility of the enantiomers and complexes containing them in the organic phase, pH and transition-metal ion (Cu) concentration. Phase-equilibria in LEXCEX systems is governed by the complex chemical equilibria in both the aqueous and organic phases. To better understand this extraction process, we develop a model for ligand-exchange chiral extraction which couples a complete description of chemical equilibria in each phase with the overall phase equilibria of the system. The model requires the complete set of protonation constants and binary and ternary formation constants for each species present in either the aqueous or organic phase. When coupled with phase equilibrium constraints, the model quantitatively predicts extraction performance as a function of key operating parameters, thereby providing a simple computational approach to process optimization. Measured equilibrium formation constants for ternary complexes containing the N-decyl-l-hydropxy-proline ligand are found to depend strongly on solvent environment, with complex stabilities in general decreasing when the complex is transferred from water to n-octanol. The role of solvent in ternary complex stability is explored through a series of molecular mechanics simulations.

The influence of solvent composition on equilibria in chiral ligand-exchange systems is explored by measuring protonation constants and equilibrium formation constants for all free species and complexes, respectively, in systems at 25°C and an ionic strength of 0.1 M containing one of two classic chiral selector molecules ( l -proline or l -hydroxy proline), the copper(II) ion, and an amino acid enantiomer. This set of thermodynamic constants is combined with multiple-chemical-equilibria theory to determine solution composition as a function of pH and mole-fraction methanol (MeOH) in the solvent. Results indicate that the addition of MeOH increases the stabilities of both the hetero- and the homo-chiral ternary complexes. However, it also increases, often to a greater extent, the stability of each bis binary complex. As a result, the influence of MeOH addition on equilibria in chiral ligand-exchange systems is complex and cannot be predicted by mere inspection of the equilibrium constants for the hetero- and homo-chiral ternary complexes. These results therefore identify a potential limitation of using common global stoichiometric models to predict equilibria in chiral ligand-exchange systems. A more comprehensive model, based on the complete description of the multiple chemical equilibria, is proposed. Finally, the role of solvent in stabilizing ligand-exchange complexes is explored through a series of molecular mechanics simulations in the absence or presence of solvent molecules. Results suggest that solvent stabilizes both bis binary and ternary complexes, primarily through direct participation in the complex structure in the form of occupancy of one or both distal coordination sites.

This paper presents a thermodynamic framework for transforming models derived in the McMillan-Mayer framework into the Gibbs framework G(T, P, ni). Historically, the McMillan-Mayer theory has been used to develop the thermodynamics of dilute solutions of electrolytes, polymers and other solutes. The appropriate independent variables for the McMillan-Mayer dilute-solution theory are temperature, volume, chemical potential of the solvent, and number of moles of solutes. Hence, the proper thermodynamic potential is not the Helmholtz energy A(T, V, nj) but a modified Helmholtz energy A′(T, V, μ0, nj (j # 0)) where A′ = A - n0μ0. The most common theoretical developments for electrolyte solutions (e.g., Debye-Huckel theory, mean-spherical approximation) and many dilute polymer solution theories (e.g., osmotic virial expansion) yield excess values of A′, not A. As a result, the chemical potential of the solute, or its activity coefficient, should include a term −P Ex V j θ , whose presence has apparently not been explicitly recognized or discussed clearly (PEx is an excess pressure, and V j θ is the partial molar volume of solute j). The origin and influence of this additional term are explored.

Excess partial molar enthalpies were measured in ternary aqueous solutions, tert -butyl alcohol (TBA)-DMSO-H 2 O, and lysozyme (L)–alcohols (A)–H 2 O. The solute–solute interactions were evaluated as the derivatives of these data with respect to the mole fraction of a solute. In the water-rich region, where the so-called mixing scheme I is operating in binary aqueous solutions of TBA, DMSO or alcohols, two solutes in TBA–DMSO–H 2 O seem to modify the hydrogen bond network of H 2 O in an additive manner. Here, mixing scheme I refers to the way in which a solute (hydrophobic) modifies the molecular organization of H 2 O. Specifically, a solute enhances the hydrogen bond strength of H 2 O in its immediate vicinity. On the other hand, the probability of hydrogen bond in the bulk away from a solute is reduced, and it is still high enough, however, to keep the hydrogen bond network connected throughout the entire macroscopic system. As the composition of solute increases, the hydrogen bond probability in the bulk decreases to the point at which the macroscopic bond connectivity is no longer possible. At this point, a new mixing scheme, II, sets in. For L–A–H 2 O mixtures at infinite dilution of L, the L–A interaction changes drastically from repulsive to attractive at the boundary between I and II for binary aqueous alcohols. Moreover, a denatured lysozyme interacts with alcohols more strongly than the native form, and is more repulsive in mixing scheme I and more attractive in II. This behaviour is shown to be consistent with the alcohol-dependent enthalpy of denaturation of lysozyme found by Velicelebi and Sturtevant [Velicelebi and Sturtevant, Biochem. 18 (1979) 1180–86].

The surface diffusion rate of bacterial cellulases from Cellulomonas fimi on cellulose was quantified using fluorescence recovery after photobleaching analysis. Studies were performed on an exo-beta-1-4-glycanase (Cex), an endo-beta-1-4-glucanase (CenA), and their respective isolated cellulose-binding domains (CBDs). Although these cellulose-binding domains bind irreversibly to microcrystalline cellulose, greater than 70% of bound molecules are mobile on the cellulose surface. Surface diffusion rates are dependent on surface coverage and range from a low of 2 x 10(-11) to a maximum of 1.2 x 10(-10) cm2/s. The fraction of mobile molecules increases only slightly with increasing fractional surface coverage density. Results demonstrate that the packing of C. fimi cellulases and their isolated binding domains onto the cellulose surface is a dynamic process. This suggests that the exclusion of potential CBD binding sites on the cellulose due to steric effects of neighboring bound CBDs may not fully explain the apparent negative cooperativity exhibited in CBD adsorption isotherms. Comparison with the kinetics of cellulase hydrolysis of crystalline substrate suggests that surface diffusion rates do not limit cellulase activity.

The adsorption of both plasmid and chromosomal duplex DNA to silica is investigated with the aim of determining the dominant forces involved in the binding reaction. Changes in the initial slopes and plateau values of adsorption isotherms for DNA on microcrystalline silica particles are used to establish the sensitivity of the binding reaction to ionic strength, temperature and pH, and DNA size and conformation. Binding is driven by an increase in entropy, with little or no enthalpic contribution. Adsorption isotherm results indicate that three effects, namely: (i) shielded intermolecular electrostatic forces, (ii) dehydration of the DNA and silica surfaces, and (iii) intermolecular hydrogen bond formation in the DNA–silica contact layer, make the dominant contributions to the overall driving force for adsorption.

Seven years have passed since one of us (W.N.) published the last comprehensive review on the mechanism of globular protein adsorption to solid/water interfaces. Since that time, annual contributions to the field have steadily increased and substantial progress has been made in a number of important areas. This review takes a fresh look at the driving force for protein adsorption by combining recent advances with key results from the past. The analysis indicates that four effects, namely structural rearrangements in the protein molecule, dehydration of (parts of) the sorbent surface, redistribution of charged groups in the interfacial layer, and protein surface polarity usually make the primary contributions to the overall adsorption behavior.