Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies

Human V_H single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human V_H domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (V_HH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human V_H single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human V_H domains and the selection of binders against a diverse set of antigens. Unlike “camelized” human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the V_H/V_L interface. Structure determination in complex with hen egg white lysozyme revealed an extended V_H binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human V_H domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies.     

A missense mutation in the agouti signaling protein gene (ASIP) is associated with the no light points coat phenotype in donkeys

Background

Seven donkey breeds are recognized by the French studbook and are characterized by a black, bay or grey coat colour including light cream-to-white points (LP). Occasionally, Normand bay donkeys give birth to dark foals that lack LP and display the no light points (NLP) pattern. This pattern is more frequent and officially recognized in American miniature donkeys. The LP (or pangare) phenotype resembles that of the light bellied agouti pattern in mouse, while the NLP pattern resembles that of the mammalian recessive black phenotype; both phenotypes are associated with the agouti signaling protein gene (ASIP).

Findings

We used a panel of 127 donkeys to identify a recessive missense c.349 T > C variant in ASIP that was shown to be in complete association with the NLP phenotype. This variant results in a cysteine to arginine substitution at position 117 in the ASIP protein. This cysteine is highly-conserved among vertebrate ASIP proteins and was previously shown by mutagenesis experiments to lie within a functional site. Altogether, our results strongly support that the identified mutation is causative of the NLP phenotype.

Conclusions

Thus, we propose to name the c.[349 T > C] allele in donkeys, the a^nlp allele, which enlarges the panel of coat colour alleles in donkeys and ASIP recessive loss-of-function alleles in animals.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0112-x) contains supplementary material, which is available to authorized users.     

A quantitative imaging-based screen reveals the exocyst as a network hub connecting endocytosis and exocytosis

The coupling of endocytosis and exocytosis underlies fundamental biological processes ranging from fertilization to neuronal activity and cellular polarity. However, the mechanisms governing the spatial organization of endocytosis and exocytosis require clarification. Using a quantitative imaging-based screen in budding yeast, we identified 89 mutants displaying defects in the localization of either one or both pathways. High-resolution single-vesicle tracking revealed that the endocytic and exocytic mutants she4∆ and bud6∆ alter post-Golgi vesicle dynamics in opposite ways. The endocytic and exocytic pathways display strong interdependence during polarity establishment while being more independent during polarity maintenance. Systems analysis identified the exocyst complex as a key network hub, rich in genetic interactions with endocytic and exocytic components. Exocyst mutants displayed altered endocytic and post-Golgi vesicle dynamics and interspersed endocytic and exocytic domains compared with control cells. These data are consistent with an important role for the exocyst in coordinating endocytosis and exocytosis.     

On-line monitoring of the transesterification reaction between triglycerides and ethanol using near infrared spectroscopy combined with gas chromatography

Many analytical procedures have been developed to determine the composition of reaction mixtures during transesterification of vegetable oils with alcohols. However, despite their accuracy, these methods are time consuming and cannot be easily used for on-line monitoring. In this work, a fast analytical method was developed to on-line monitor the transesterification reaction of high oleic sunflower oil with ethanol using Near InfraRed spectroscopy and a multivariate approach. The reactions were monitored through sequential scans of the reaction medium with a probe in a one-liter batch reactor without collecting and preparing samples. To calibrate the NIR analytical method, gas chromatography-flame ionization detection was used as a reference method. The method was validated by studying the kinetics of the EtONa-catalyzed transesterification reaction. Activation energy (51.0 kJ/mol) was also determined by considering a pseudo second order kinetics model.     

Synthesis and SAR of novel quinazolines as potent and brain-penetrant c-jun N-terminal kinase (JNK) inhibitors

Quinazoline 3 was discovered as a novel c-jun N-terminal kinase (JNK) inhibitor with good brain penetration and pharmacokinetic (PK) properties. A number of analogs which were potent both in the biochemical and cellular assays were discovered. Quinazoline 13a was found to be a potent JNK3 inhibitor (IC₅₀ = 40 nM), with >500-fold selectivity over p38, and had good PK and brain penetration properties. With these properties, 13a is considered a potential candidate for in vivo evaluation.