Increased yields and biological potency of knob-into-hole-based soluble MHC class II molecules.

Assembly of soluble peptide-major histocompatibility complex class II (pMHCII) monomers into multimeric structures enables the detection of antigen-specific CD4+ T cells in biological samples and, in some configurations, their reprogramming in vivo. Unfortunately, current MHCII-αß chain heterodimerization strategies are typically associated with low production yields and require the use of foreign affinity tags for purification, precluding therapeutic applications in humans. Here, we show that fusion of peptide-tethered or empty MHCII-αß chains to the IgG1-Fc mutated to form knob-into-hole structures results in the assembly of highly stable pMHCII monomers. This design enables the expression and rapid purification of challenging pMHCII types at high yields without the need for leucine zippers and purification affinity tags. Importantly, this design increases the antigen-receptor signaling potency of multimerized derivatives useful for therapeutic applications and facilitates the detection and amplification of low-avidity T cell specificities in biological samples using flow cytometry.

A simple technique based on a single optical trap for the determination of bacterial swimming pattern.

Bacterial motility is associated to a wide range of biological processes and it plays a key role in the virulence of many pathogens. Here we describe a method to distinguish the dynamic properties of bacteria by analyzing the statistical functions derived from the trajectories of a bacterium trapped by a single optical beam. The approach is based on the model of the rotation of a solid optically trapped sphere. The technique is easily implemented in a biological laboratory, since with only a small number of optical and electronic components a simple biological microscope can be converted into the required analyzer. To illustrate the functionality of this method, we probed several Salmonella enterica serovar Typhimurium mutants that differed from the wild-type with respect to their swimming patterns. In a further application, the motility dynamics of the S. Typhimurium cheV mutant were characterized.