Online comprehensive two-dimensional liquid chromatography (online LC x LC) has become increasingly popular. Among the different chromatographic modes that can be combined, hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) are particularly interesting because they offer a high degree of orthogonality. However, this combination remains complex due to the incompatibility of the solvents in the two dimensions. To avoid this problem, it is possible to dilute the first dimension (1D) effluent with (zdilution -1) volumes of a weaker solvent added to one volume of 1D-effluent, where zdilution represents the extent to which the fraction volume has been multiplied. This can be done using either active solvent modulation technology or an additional pump, prior to the second dimension analysis. The objective of this study was to develop theoretical models to predict whether or not dilution can be effective, and, if so, what is the minimum zdilution value required. This approach is based on the calculation of the ratio (called xdilution) between the peak standard deviation due to the injection process and the peak standard deviation in the absence of extra-column dispersion. xdilution was calculated from theoretical relationships and plotted as a function of zdilution, to predict the value required to obtain good peak shapes for the compound of interest. The maximum xdilution value was found to be of the order of 1 for chromatographically acceptable peak shapes. The proposed theoretical approach was experimentally validated on a number of representative small molecules and peptides. Agreement between experimental results and theoretical models was very high, especially for small molecules. Finally, it is shown that this approach helps to predict the most appropriate set of conditions in HILIC x RPLC, depending on the compounds to be separated.

Multifunctional nanocarriers enhance the treatment efficacy for modern therapeutics and have gained increasing importance in biomedical research. Codelivery of multiple bioactive molecules enables synergistic therapies. Coencapsulation of cargo molecules into one nanocarrier system is challenging due to different physicochemical properties of the cargo molecules. Additionally, coencapsulation of multiple molecules simultaneously shall proceed with high control and efficiency. Orthogonal approaches for the preparation of nanocarriers are essential to encapsulate sensitive bioactive molecules while preserving their bioactivity. Preparation of nanocarriers by physical processes (i.e., self-assembly or coacervation) and chemical reactions (i.e., click reactions, polymerizations, etc.) are considered as orthogonal methods to most cargo molecules. This review shall act as a guideline to allow the reader to select a suitable preparation protocol for a desired nanocarrier system. This article helps to select for combinations of cargo molecules (hydrophilic-hydrophobic, small-macro, organic-inorganic) with nanocarrier material and synthesis protocols. The focus of this article lies on the coencapsulation of multiple cargo molecules into biocompatible and biodegradable nanocarriers prepared by orthogonal strategies. With this toolbox, the selection of a preparation method for a known set of cargo molecules to prepare the desired biodegradable and loaded nanocarrier shall be provided.

The Organisation for Economic Co-operation and Development guideline 305 for bioaccumulation testing in fish includes the option to conduct a dietary test for assessing a chemical\'s bioaccumulation behavior. However, the one-compartment toxicokinetic model that is used in the guidelines to analyze the results from dietary bioaccumulation tests is not consistent with the current state of the science, experimental practices, and information needs for bioaccumulation and risk assessment. The present study presents 1) a 2-compartment toxicokinetic modeling framework for describing the bioaccumulation of neutral hydrophobic organic chemicals in fish and 2) an associated toxicokinetic analysis tool (absorption, distribution, metabolism, and excretion [ADME] B calculator) for the analysis and interpretation of dietary bioaccumulation test data from OECD-305 dietary tests. The model framework and ADME-B calculator are illustrated by analysis of fish dietary bioaccumulation test data for 238 substances representing different structural classes and susceptibilities to biotransformation. The ADME of the chemicals is determined from dietary bioaccumulation tests and bioconcentration factors, biomagnification factors, and somatic and intestinal biotransformation rates. The 2-compartment fish toxicokinetic model can account for the effect of the exposure pathway on bioaccumulation, which the one-compartment model cannot. This insight is important for applying a weight-of-evidence approach to bioaccumulation assessment where information from aqueous and dietary test endpoints can be integrated to improve the evaluation of a chemical\'s bioaccumulation potential. Environ Toxicol Chem 2019;39:171-188. © 2019 SETAC.

This work describes a novel strategy for the integrated expression and purification of recombinant proteins in Pichia pastoris cultures. Hydrophobins can be used as fusion tags, proteins fused to them alter their hydrophobicity and can be purified by aqueous two-phase systems (ATPS) based on non-ionic surfactants. Here, the consensus dengue virus envelope protein domain III fused to hydrophobin I of Trichoderma reesei was expressed in Pichia pastoris cultures and an in situ product removal by an ATPS using a non-ionic detergent, (Triton X-114) was performed. The protein was produced and purified directly from the yeast culture supernatant both efficiently and with no loss. The purified protein was properly immobilized by adsorption in solid phase and recognized by anti-dengue antibodies, showing its potential for the development of an indirect immunoassay for dengue virus.

Eight software applications are compared for their performance in estimating the octanol-water partition coefficient (Kow), melting point, vapor pressure and water solubility for a dataset of polychlorinated biphenyls, polybrominated diphenyl ethers, polychlorinated dibenzodioxins, and polycyclic aromatic hydrocarbons. The predicted property values are compared against a curated dataset of measured property values compiled from the scientific literature with careful consideration given to the analytical methods used for property measurements of these hydrophobic chemicals. The variability in the predicted values from different calculators generally increases for higher values of Kow and melting point and for lower values of water solubility and vapor pressure. For each property, no individual calculator outperforms the others for all four of the chemical classes included in the analysis. Because calculator performance varies based on chemical class and property value, the geometric mean and the median of the calculated values from multiple calculators that use different estimation algorithms are recommended as more reliable estimates of the property value than the value from any single calculator.

The challenge in achieving precision medicine relies on how to advance and/or enhance new as well as old therapeutic strategies. Here, we highlight the significant role hydrophilicity of biotinylated fluorescent probe\'s plays on their cellular uptake behaviour.

In an attempt to seek increased understanding of compound attributes that influence successful drug pipeline progression, GlaxoSmithKline\'s portfolio of oral candidates was compared with reference sets of marketed oral drugs. The approach differs from other attrition studies by explicitly focusing on choosing \'the right compound\' by applying relevant, experimentally derived properties. The analysis led to four proposed compound quality categories, created by combining specific criteria for three measures: dose, solubility and the property forecast index, a composite measure of lipophilicity using chromatographically determined LogD and aromaticity. The \'three properties\' provide benchmarked guidelines for project teams to use when seeking and selecting clinical candidates, because they reflect the property distribution of marketed oral drugs.

Peptide coatings on material surfaces have demonstrated wide application across materials science and biotechnology, facilitating the development of nanobio interfaces through surface modification. A guiding motivation in the field is to engineer peptides with a high and selective binding affinity to target materials. Herein, we introduce a quantitative force mapping method in order to evaluate the binding affinity of peptides to various hydrophilic oxide materials by atomic force microscopy (AFM). Statistical analysis of adhesion forces and probabilities obtained on substrates with a materials contrast enabled us to simultaneously compare the peptide binding affinity to different materials. On the basis of the experimental results and corresponding theoretical analysis, we discuss the role of various interfacial forces in modulating the strength of peptide attachment to hydrophilic oxide solid supports as well as to gold. The results emphasize the precision and robustness of our approach to evaluating the adhesion strength of peptides to solid supports, thereby offering guidelines to improve the design and fabrication of peptide-coated materials.

Asparagine (N)-linked glycosylation is essential for efficient protein folding in the endoplasmic reticulum (ER) and anterograde trafficking through the secretory pathway. N-Glycans are attached to nascent polypeptides at consensus sites, N-X-T/S (X ≠ P), by one of two enzymatic isoforms of the oligosaccharyltransferase (OST), STT3A or STT3B. Here, we examined the effect of the consensus site X and hydroxyl residue on the distributions of co- and post-translational N-glycosylation of a type I transmembrane glycopeptide scaffold. Using rapid radioactive pulse-chase experiments to resolve co-translational (STT3A) and post-translational (STT3B) events, we determined that NXS consensus sites containing large hydrophobic and negatively charged middle residues are frequently skipped by STT3A during protein translation. Post-translational modification of the cotranslationally skipped sites by STT3B was similarly hindered by the middle X residue, resulting in hypoglycosylation of NXS sites containing large hydrophobic and negatively charged side chains. In contrast, NXT consensus sites (barring NWT) were efficiently modified by the cotranslational machinery, reducing STT3B\'s role in modifying consensus sites skipped during protein translation. A strong correlation between cotranslational N-glycosylation efficiency and the rate of post-translational N-glycosylation was determined, showing that the OST STT3A and STT3B isoforms are similarly influenced by the hydroxyl and middle X consensus site residues. Substituting various middle X residues into an OST eubacterial homologous structure revealed that small and polar consensus site X residues fit well in the peptide binding site whereas large hydrophobic and negatively charged residues were harder to accommodate, indicating conserved enzymatic mechanisms for the mammalian OST isoforms.

The crystal structure of an N-terminal β-strand-swapped consensus-derived tenascin FN3 alternative scaffold has been determined. A comparison with the unswapped structure reveals that the side chain of residue F88 orients differently and packs more tightly with the hydrophobic core of the domain. Dimer formation also results in the burial of a hydrophobic patch on the surface of the domain. Thus, it appears that tighter packing of F88 in the hydrophobic core and burial of surface hydrophobicity provide the driving forces for the N-terminal β-strand swapping, leading to the formation of a stable compact dimer.