Bacterial Communities Vary between Sinuses in Chronic Rhinosinusitis Patients.

Chronic rhinosinusitis (CRS) is a common and potentially debilitating disease characterized by inflammation of the sinus mucosa for longer than 12 weeks. Bacterial colonization of the sinuses and its role in the pathogenesis of this disease is an ongoing area of research. Recent advances in culture-independent molecular techniques for bacterial identification have the potential to provide a more accurate and complete assessment of the sinus microbiome, however there is little concordance in results between studies, possibly due to differences in the sampling location and techniques. This study aimed to determine whether the microbial communities from one sinus could be considered representative of all sinuses, and examine differences between two commonly used methods for sample collection, swabs, and tissue biopsies. High-throughput DNA sequencing of the bacterial 16S rRNA gene was applied to both swab and tissue samples from multiple sinuses of 19 patients undergoing surgery for treatment of CRS. Results from swabs and tissue biopsies showed a high degree of similarity, indicating that swabbing is sufficient to recover the microbial community from the sinuses. Microbial communities from different sinuses within individual patients differed to varying degrees, demonstrating that it is possible for distinct microbiomes to exist simultaneously in different sinuses of the same patient. The sequencing results correlated well with culture-based pathogen identification conducted in parallel, although the culturing missed many species detected by sequencing. This finding has implications for future research into the sinus microbiome, which should take this heterogeneity into account by sampling patients from more than one sinus.

Chilling-induced ultrastructural changes to mesophyll cells of Arabidopsis grown under short days are almost completely reversible by plant re-warming.

Exposure of plants to chilling (low temperatures above freezing) limits growth and development in all environments outside the lowest latitudes. Cell ultrastructure and morphometric studies may allow associations to be made between chilling-induced changes at the ultrastructural level, molecular events and their physiological consequences. We examined changes in the shape, size and membrane organization of the organelles of mesophyll cells in Arabidopsis thaliana (Col 0), a cold-resistant species, after subjecting 6-week-old plants grown at normal growth temperatures to chilling (2.5-4°C; 14-h dark/10-h light cycle) for 6, 24 and 72 h and after a re-warming period of 50 h. No ultrastructural differences were seen in the first 6 h of chilling but after 24 h we observed swollen and rounded chloroplasts with larger starch grains and dilated thylakoids compared to control plants. By 72 h, chilling had resulted in a large accumulation of starch in chloroplasts, an apparent crowding of the cytosol and a lower abundance of peripheral reticulum than in the controls. The average area per chloroplast in cell sections increased after 72-h chilling while the number of chloroplasts remained the same. Ring-shaped and other morphologically aberrant mitochondria were present in significantly higher abundance in plants given 72 h chilling than in the controls. Plant re-warming for 50 h reduced chloroplast size to those of the controls and returned mitochondria to standard morphology, but peripheral reticulum remained less abundant than in plants never given a cold treatment. The near full return to normal ultrastructure upon plant re-warming indicates that the morphological changes may be part of acclimation to cold.

Arabidopsis AtSerpin1, crystal structure and in vivo interaction with its target protease RESPONSIVE TO DESICCATION-21 (RD21).

In animals, protease inhibitors of the serpin family are associated with many physiological processes, including blood coagulation and innate immunity. Serpins feature a reactive center loop (RCL), which displays a protease target sequence as a bait. RCL cleavage results in an irreversible, covalent serpin-protease complex. AtSerpin1 is an Arabidopsis protease inhibitor that is expressed ubiquitously throughout the plant. The x-ray crystal structure of recombinant AtSerpin1 in its native stressed conformation was determined at 2.2 A. The electrostatic surface potential below the RCL was found to be highly positive, whereas the breach region critical for RCL insertion is an unusually open structure. AtSerpin1 accumulates in plants as a full-length and a cleaved form. Fractionation of seedling extracts by nonreducing SDS-PAGE revealed the presence of an additional slower migrating complex that was absent when leaves were treated with the specific cysteine protease inhibitor L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane. Significantly, RESPONSIVE TO DESICCATION-21 (RD21) was the major protease labeled with the L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane derivative DCG-04 in wild type extracts but not in extracts of mutant plants constitutively overexpressing AtSerpin1, indicating competition. Fractionation by nonreducing SDS-PAGE followed by immunoblotting with RD21-specific antibody revealed that the protease accumulated both as a free enzyme and in a complex with AtSerpin1. Importantly, both RD21 and AtSerpin1 knock-out mutants lacked the serpin-protease complex. The results establish that the major Arabidopsis plant serpin interacts with RD21. This is the first report of the structure and in vivo interaction of a plant serpin with its target protease.