Fidgetin interacting with microtubule end binding protein EB3 affects axonal regrowth in spinal cord injury

Fidgetin,a microtubule-severing enzyme,regulates neurite outgrowth,axonal regeneration,and cell migration by trimming off the labile domain of microtubule polymers.Because maintenance of the microtubule labile domain is essential for axon initiation,elongation,and navigation,it is of interest to determine whether augmenting the microtubule labile domain via depletion of fidgetin serves as a therapeutic approach to promote axonal regrowth in spinal cord injury.In this study,we constructed rat models of spinal cord injury and sciatic nerve injury.Compared with spinal cord injury,we found that expression level of tyrosinated microtubules in the labile portion of microtubules continuously increased,whereas fidgetin decreased after peripheral nerve injury.Depletion of fidgetin enhanced axon regeneration after spinal cord injury,whereas expression level of end binding protein 3(EB3)markedly increased.Next,we performed RNA interference to knockdown EB3 or fidgetin.We found that deletion of EB3 did not change fidgetin expression.Conversely,deletion of fidgetin markedly increased expression of tyrosinated microtubules and EB3.Deletion of fidgetin increased the amount of EB3 at the end of neurites and thereby increased the level of tyrosinated microtubules.Finally,we deleted EB3 and overexpressed fidgetin.We found that fidgetin trimmed tyrosinated tubulins by interacting with EB3.When fidgetin was deleted,the labile portion of microtubules was elongated,and as a result the length of axons and number of axon branches were increased.These findings suggest that fidgetin can be used as a novel therapeutic target to promote axonal regeneration after spinal cord injury.Furthermore,they reveal an innovative mechanism by which fidgetin preferentially severs labile microtubules.

Optimization on Ultrasonic Extraction Method of Total Flavonoids for Liriodendron chinense Sarg.×L.tulipifera L. by Response Surface Methodology (RSM)

With the leaves,bark and roots of Liriodendron chinense Sarg.× L.tulipifera L.as experiment materials,the ultrasonic-assisted extraction conditions of total flavonoids were optimized by response surface Box-Behnken test design.Ultrasonic time and liquid-to-solid ratio had significant effects on the extraction amount of flavonoids from the leaves of L.chinense Sarg.× L.tulipifera L.;ultrasonic temperature and liquid-to-solid ratio had an interaction on the extraction amount of flavonoids;and the optimum ultrasonic time,temperature and liquid-to-solid ratio for the extraction of flavonoids from leaves of L.chinense Sarg.× L.tulipifera L.were 19.82 min,28.60 ℃ and 9.48 ml/g,respectively.Ultrasonic time,temperature and liquid-to-solid ratio had significant effects on the extraction amount of flavonoids from L.chinense Sarg.× L.tulipifera L.;there was a significant interaction between any two of the factors,which had a significant effect on the extraction of flavonoids;and the optimum extraction parameters for flavonoids in the bark were the ultrasonic time of 33.66 min,the ultrasonic temperature at 32.85 ℃ and the liquid-to-solid ratio of 11.39 ml/g.Ultrasonic time and liquid-to-solid ratio had significant effects on the extraction of flavonoids from roots;there was a significant interaction between ultrasonic time and liquid-to-solid ratio and between ultrasonic temperature and liquid-to-solid ratio;and the optimum extraction parameters for flavonoids from the roots of L.chinense Sarg.× L.tulipifera L.were the ultrasonic time of 32.38 min,the ultrasonic temperature at 25.0 ℃ and the liquid-to-solid ratio of 8.00 ml/g.The results of the three models were stable.The models are feasible and have good application value.