Biosynthesis and differential spatial distribution of the 3-deoxyanthocyanidins apigenidin and luteolinidin at the interface of a plant-cyanobacteria symbiosis exposed to cold.

Aquatic ferns of the genus Azolla (Azolla) form highly productive symbioses with filamentous cyanobacteria fixing N2 in their leaf cavities, Nostoc azollae. Stressed symbioses characteristically turn red due to 3-deoxyanthocyanidin (DA) accumulation, rare in angiosperms and of unknown function. To understand DA accumulation upon cold acclimation and recovery, we integrated laser-desorption-ionization mass-spectrometry-imaging (LDI-MSI), a new Azolla filiculoides genome-assembly and annotation, and dual RNA-sequencing into phenotypic analyses of the symbioses. Azolla sp. Anzali recovered even when cold-induced DA-accumulation was inhibited by abscisic acid. Cyanobacterial filaments generally disappeared upon cold acclimation and Nostoc azollae transcript profiles were unlike those of resting stages formed in cold-resistant sporocarps, yet filaments re-appeared in leaf cavities of newly formed green fronds upon cold-recovery. The high transcript accumulation upon cold acclimation of AfDFR1 encoding a flavanone 4-reductase active in vitro suggested that the enzyme of the first step in the DA-pathway may regulate accumulation of DAs in different tissues. However, LDI-MSI highlighted the necessity to describe metabolite accumulation beyond class assignments as individual DA and caffeoylquinic acid metabolites accumulated differentially. For example, luteolinidin accumulated in epithelial cells, including those lining the leaf cavity, supporting a role for the former in the symbiotic interaction during cold acclimation.

Influence of Ion Source Geometry on the Repeatability of Topographically Guided LAESI-MSI.

Spatially resolving the relative distribution of analyte molecules in biological matter holds great promise in the life sciences. Mass spectrometry imaging (MSI) is a technique that can provide such spatial resolution but remains underused in fields such as chemical ecology, as traditional MSI sample preparation is often chemically or morphologically invasive. Laser ablation electrospray ionization (LAESI)-MSI is a variation of MSI particularly well-suited for situations where chemical sample preparation is too invasive but provides new challenges related to the repeatability of measurement outcomes. We assess the repeatability of LAESI-MSI by sampling a droplet of [ring-13C6]l-phenylalanine with known concentration and expressing the resulting variability as a coefficient of variation, cv. In doing so, we entirely eliminate variability caused by surface morphology or underlying true sample gradients. We determine the limit of detection (LOD) for13C6-Phe by sampling from droplets with successively decreasing but known concentration. We assess the influence of source geometry on the LOD and repeatability by performing these experiments using four distinct variations of sources: one commercial and three custom-built ones. Finally, we extend our study to leaf and stem samples Arabidopsis thaliana and Gossypium hirsutum. We overcome the challenges of LAESI associated with three-dimensional surface morphology by relying on work previously published. Our measurements on both controlled standard and realistic samples give strong evidence that LAESI-MSI's repeatability in current implementations is insufficient for MSI in chemical ecology.

Motility, Chemotaxis and Aerotaxis Contribute to Competitiveness during Bacterial Pellicle Biofilm Development.

Biofilm formation is a complex process involving various signaling pathways and changes in gene expression. Many of the sensory mechanisms and regulatory cascades involved have been defined for biofilms formed by diverse organisms attached to solid surfaces. By comparison, our knowledge on the basic mechanisms underlying the formation of biofilms at air-liquid interfaces, that is, pellicles, is much less complete. In particular, the roles of flagella have been studied in multiple solid-surface biofilm models but remain largely undefined for pellicles. In this work, we characterize the contributions of flagellum-based motility, chemotaxis and oxygen sensing to pellicle formation in the Gram-positive Bacillus subtilis. We confirm that flagellum-based motility is involved in, but is not absolutely essential for, B. subtilis pellicle formation. Further, we show that flagellum-based motility, chemotaxis and oxygen sensing are important for successful competition during B. subtilis pellicle formation. We report that flagellum-based motility similarly contributes to pellicle formation and fitness in competition assays in the Gram-negative Pseudomonas aeruginosa. Time-lapse imaging of static liquid cultures demonstrates that, in both B. subtilis and P. aeruginosa, a turbulent flow forms in the tube and a zone of clearing appears below the air-liquid interface just before the formation of the pellicle but only in strains that have flagella.

Spatially resolved in vivo plant metabolomics by laser ablation-based mass spectrometry imaging (MSI) techniques: LDI-MSI and LAESI.

This short review aims to summarize the current developments and applications of mass spectrometry-based methods for in situ profiling and imaging of plants with minimal or no sample pre-treatment or manipulation. Infrared-laser ablation electrospray ionization and UV-laser desorption/ionization methods are reviewed. The underlying mechanisms of the ionization techniques-namely, laser ablation of biological samples and electrospray ionization-as well as variations of the LAESI ion source for specific targets of interest are described.

Biomechanical stability of an arthroscopic anterior capsular shift and suture anchor repair in anterior shoulder instability: a human cadaveric shoulder model.

It was hypothesized that an arthroscopic Bankart repair with suture anchors supplies sufficient anterior shoulder stability, which cannot be improved by an additional capsular shift. In an experimental biomechanical human cadaver study, we tested ten fresh human cadaver shoulders in a robot-assisted shoulder simulator. External rotation and glenohumeral translation were measured at 0 degrees and 80 degrees of glenohumeral abduction. All measurements were performed under the following conditions: on the non-operated shoulder; following the setting of three arthroscopic portals; following an arthroscopic anterior capsular shift; following a simulated Bankart lesion; and following an arthroscopic Bankart repair. The application of three arthroscopic portals resulted in a significant increase of the anterior (P = 0.01) and antero-inferior translation (P = 0.03) at 0 degrees and 80 degrees abduction, as well as an increase in external rotation at 80 degrees abduction (P = 0.03). Capsular shift reduced external rotation (P = 0.03), but did not significantly decrease translation. Simulating anterior shoulder instability, glenohumeral translation significantly increased, ranging from 50 to 279% of physiological translation. Arthroscopic shoulder stabilization resulted in a decrease of translation in all tested directions to approximately physiologic levels. External rotation in 0 degrees abduction was thus decreased significantly (P = 0.003) to an average of 19 degrees . The study proved that an arthroscopic anterior capsular shift in a cadaveric model decreases external rotation without a significant influence on glenohumeral translation. Arthroscopic shoulder stabilization with suture anchors thus sufficiently restores increased glenohumeral translation, but also decreases external rotation in neutral abduction. An anatomic reconstruction of the Bankart lesion without overconstraining of the antero-inferior capsule should therefore be the aim in arthroscopic anterior shoulder stabilization.