Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution.

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric complexes. They interact with other cytoskeletal components and the cell membrane to participate in important cellular functions such as migration and cell division. Due to the complexity of septins' many interactions, the large number of septin genes (13 in humans), and the ability of septins to form hetero-oligomeric complexes with different subunit compositions, cell-free reconstitution is a vital strategy to understand the basics of septin biology. The present paper first describes a method to purify recombinant septins in their hetero-oligomeric form using a two-step affinity chromatography approach. Then, the process of quality control used to check for the purity and integrity of the septin complexes is detailed. This process combines native and denaturing gel electrophoresis, negative stain electron microscopy, and interferometric scattering microscopy. Finally, a description of the process to check for the polymerization ability of septin complexes using negative stain electron microscopy and fluorescent microscopy is given. This demonstrates that it is possible to produce high-quality human septin hexamers and octamers containing different isoforms of septin_9, as well as Drosophila septin hexamers.

A new portable sampler to monitor pollen at street level in the environment of patients.

Allergic rhinitis caused by pollen exposure is one of the most common allergic diseases. Therefore monitoring pollen levels in ambient air is an important tool in research and health care. Most European monitoring stations collect airborne pollen at rooftop levels for measurements in the larger surrounding of the sampling station, and not in the direct environment of sensitized subjects. Here we present the development and evaluation of a portable pollen sampler, called "Pollensniffer", that was designed to collect pollen in the immediate environment of allergic subjects. Validation of the Pollensniffer against the standard volumetric pollen sampler showed for most pollen types high correlations between the number of pollen collected by those two devices (Spearman's Correlation Coefficient > 0.8); the Pollensniffer appeared to collect on average 5.8 times more pollen per hour than the static sampler. Pollen monitoring was performed using this Pollensniffer at street level at 3 different locations in the city of Leiden during 22 weeks in 2017 and 21 weeks in 2018, during three 15-min periods a day and at one day in the week. The results showed that the pollen levels for birch and grass pollen can significantly differ from location to location and per time of day. Furthermore, the Pollensniffer measurements at street level showed that birch and grass pollen grains were detected 1 1/2 and 2-3 weeks, respectively, before detection at rooftop level. The street measurements show that allergic subjects can encounter varying pollen levels throughout the city and that they can be exposed to grass and birch pollen and may experience hay fever symptoms, even before the sampler at rooftop level registers these pollen.