No abstract

Traumatic strain injury in skeletal muscle is often associated with fluid accumulation at the site of rupture, but the role of this injury exudate (EX) in cellular responses and healing is unknown. We aimed to characterize the EX sampled from human hamstring or calf muscles following a strain injury (n = 12). The cytokine and growth‐factor profile, gene expression, and transcriptome analysis of EX‐derived cells were compared with blood taken simultaneously from the same individuals. Cellular responses to the EX were tested in 3‐dimensional (3D) culture based on primary human fibroblasts and myoblasts isolated from hamstring muscles. The EX contained a highly proinflammatory profile with a substantial expression of angiogenic factors. The proinflammatory profile was present in samples taken early postinjury and in samples aspirated several weeks postinjury, suggesting persistent inflammation. Cells derived from the EX demonstrated an increased expression of fibrogenic, adipogenic, and angiogenesis‐related genes in comparison with blood cells. The injury EX stimulated fibroblast proliferation 2‐fold compared with plasma, whereas such an effect was not seen for myoblasts. Finally, in 3D cell culture, the EX induced an up‐regulation of connective tissue‐related genes. In summary, EX formation following a muscle‐strain injury stimulates fibroblast proliferation and the synthesis of connective tissue in fibroblasts. This suggests that the EX promotes an acute tissue‐healing response but potentially also contributes to the formation of fibrotic tissue in the later phases of tissue repair.—Bayer, M. L., Bang, L., Hoegberget‐Kalisz, M., Svensson, R. B., Olesen, J. L., Karlsson, M. M., Schjerling, P., Hellsten, Y., Hoier, B., Magnusson, S. P., Kjaer, M. Muscle‐strain injury exudate favors acute tissue healing and prolonged connective tissue formation in humans. FASEB J. 33,10369–10382 (2019). http://www.fasebj.org

When positioned horizontally, roots grow down toward the direction of gravity. This phenomenon, called gravitropism, is influenced by most of the major plant hormones including brassinosteroids. Epi-brassinolide (eBL) was previously shown to enhance root gravitropism, a phenomenon similar to the response of roots exposed to the actin inhibitor, latrunculin B (LatB). This led us to hypothesize that eBL might enhance root gravitropism through its effects on filamentous-actin (F-actin). This hypothesis was tested by comparing gravitropic responses of maize (Zea mays) roots treated with eBL or LatB. LatB-and eBL-treated roots displayed similar enhanced downward growth compared with controls when vertical roots were oriented horizontally. Moreover, the effects of the two compounds on root growth directionality were more striking on a slowly-rotating twodimensional clinostat. Both compounds inhibited autotropism, a process in which the root straightened after the initial gravistimulus was withdrawn by clinorotation. Although eBL reduced F-actin density in chemically-fixed Z. mays roots, the impact was not as strong as that of LatB. Modification of F-actin organization after treatment with both compounds was also observed in living roots of barrel medic (Medicago truncatula) seedlings expressing genetically encoded F-actin reporters. Like in fixed Z. mays roots, eBL effects on F-actin in living M. truncatula roots were modest compared with those of LatB. Furthermore, live cell imaging revealed a decrease in global F-actin dynamics in hypocotyls of etiolated M. truncatula seedlings treated with eBL compared to controls. Collectively, our data indicate that eBL-and LatB-induced enhancement of root gravitropism can be explained by inhibited autotropic root straightening, and that eBL affects this process, in part, by modifying F-actin organization and dynamics.

TILLING is a low-cost screening method that allows for identification of mutations in a gene-of-interest within a range of few base pairs. TILLING can be applied to mutant populations or to plant collections of cultivars, landraces or crop wild relatives (Eco-TILLING). The method is based on the Cel1 enzyme cleavage of mismatches in PCR products amplified with labeled primers. The cleavage can be detected due to the labeled primers by different methods including capillary electrophoresis. Here, we introduce the development of the mutant population BRACHYLIFE and present a Brachypodium TILLING protocol based on fluorescing primers for PCR, enzymatic cleavage, and detection with Applied Biosystems 3130xl Genetic Analyzer.