Editing of endogenous tubulins reveals varying effects of tubulin posttranslational modifications on axonal growth and regeneration.

Tubulin posttranslational modifications (PTMs) modulate the dynamic properties of microtubules and their interactions with other proteins. However, the effects of tubulin PTMs were often revealed indirectly through the deletion of modifying enzymes or the overexpression of tubulin mutants. In this study, we directly edited the endogenous tubulin loci to install PTM-mimicking or -disabling mutations and studied their effects on microtubule stability, neurite outgrowth, axonal regeneration, cargo transport, and sensory functions in the touch receptor neurons of Caenorhabditis elegans. We found that the status of ß-tubulin S172 phosphorylation and K252 acetylation strongly affected microtubule dynamics, neurite growth, and regeneration, whereas α-tubulin K40 acetylation had little influence. Polyglutamylation and detyrosination in the tubulin C-terminal tail had more subtle effects on microtubule stability likely by modulating the interaction with kinesin-13. Overall, our study systematically assessed and compared several tubulin PTMs for their impacts on neuronal differentiation and regeneration and established an in vivo platform to test the function of tubulin PTMs in neurons.

[Effects of harvest residue management on soil organic nitrogen fractions in young Cunninghamia lanceolata plantation]. / 采伐剩余物不同处理方式对杉木幼林土壤有机氮组分的影响.

Different treatments of harvest residues will change the quantity and quality of soil organic matter, with direct or indirect effects on the composition and content of soil nutrient. Nitrogen is one of the most important soil nutrients. However, the response of soil organic nitrogen fractions to different harvest residue treatments is still unclear. In this study, harvest residue treatments, including harvest residue removed, residue retained and residue burnt, were set up after clear-cutting a 50-year-old mature Cunninghamia lanceolata forest in Sanming City, Fujian, China. The H2SO4 hydrolysis method was used to determine soil organic nitrogen fractions and their driving factors in the 0-10 cm and 10-20 cm soil layers after 5 years of harvest residue treatments. The results showed that residue retained treatment significantly enhanced the contents of soil organic nitrogen and its liable fractions. In the 0-10 cm soil layer, soil organic nitrogen content under residue retained treatment (3.36 g·kg-1) was 1.5 and 1.3 times as those of residue removed and residue burnt treatments, respectively. Residue retained treatment had the highest contents of labile nitrogen Ⅰ and Ⅱ fractions. In 10-20 cm soil layer, the contents of soil organic nitrogen and labile nitrogen Ⅱ fraction were also significantly higher in residue retained treatment (2.20, 0.73 g·kg-1) than that in residue removed and residue burnt treatments. The labile nitrogen index Ⅱ in residue retained treatment (33.9%) was significantly higher than in residue burnt treatment (26.1%). The contents of total carbon, dissolved organic carbon, dissolved organic nitrogen, microbial biomass under residue retained treatment were the highest in both soil layers. Compared with residue removed treatment, residue retained treatment significantly enhanced the abundance of soil bacteria (Gram-positive bacteria and Gram-negative bacteria) in 0-10 cm soil layer. In 10-20 cm soil layer, residue retained treatment had the highest content of fungi and the lowest content of actinomycetes. Pearson analysis showed that there were significant positive correlations of labile fractions of soil organic nitrogen with total carbon, dissolved organic carbon, dissolved organic nitrogen, microbial biomass carbon, microbial biomass nitrogen, bacteria (Gram-positive bacteria, Gram-negative bacteria), and fungi, and negative correlations with actinomycetes. It was concluded that the retention of harvest residue was beneficial to increase the content of soil organic nitrogen and labile fractions, improve soil biochemical properties and had a positive effect on soil microbial community composition. Retention of harvest residue was an effective management measure to maintain soil fertility and improve forest productivity.

The Expression and Function of Tubulin Isotypes in Caenorhabditis elegans.

Microtubules, made from the polymerization of the highly conserved α/ß-tubulin heterodimers, serve as important components of the cytoskeleton in all eukaryotic cells. The existence of multiple tubulin isotypes in metazoan genomes and a dazzling variety of tubulin posttranslational modifications (PTMs) prompted the "tubulin code" hypothesis, which proposed that microtubule structure and functions are determined by the tubulin composition and PTMs. Evidence for the tubulin code has emerged from studies in several organisms with the characterization of specific tubulins for their expression and functions. The studies of tubulin PTMs are accelerated by the discovery of the enzymes that add or remove the PTMs. In tubulin research, the use of simple organisms, such as Caenorhabditis elegans, has been instrumental for understanding the expression and functional specialization of tubulin isotypes and the effects of their PTMs. In this review, we summarize the current understanding of the expression patterns and cellular functions of the nine α-tubulin and six ß-tubulin isotypes. Expression studies are greatly facilitated by the CRISPR/Cas9-mediated endogenous GFP knock-in reporters and the organism-wide single cell transcriptomic studies. Meanwhile, functional studies benefit from the ease of genetic manipulation and precise gene replacement in C. elegans. These studies identified both ubiquitously expressed tubulin isotypes and tissue-specific isotypes. The isotypes showed functional redundancy, as well as functional specificity, which is likely caused by the subtle differences in their amino acid sequences. Many of these differences concentrate at the C-terminal tails that are subjected to several PTMs. Indeed, tubulin PTM, such as polyglutamylation, is shown to modulate microtubule organization and properties in both ciliated and non-ciliated neurons. Overall, studies from C. elegans support the distinct expression and function patterns of tubulin isotypes and the importance of their PTMs and offer the promise of cracking the tubulin code at the whole-genome and the whole-organism level.

[Experimental study of repairing femoral bone defects with nHA/RHLC/PLA scaffold composite with endothelial cells and osteoblasts in canines].

OBJECTIVE: To explore whether a tissue-engineered construct composed of autogenous endothelial cells, osteoblasts and a new bioresorbable nano-hydroxyapatite/recombinant human-like collagen/polylactic acid (nHA/RHLC/PLA) would enhance bone regeneration and repair femoral head defects in canine models. METHODS: The bone marrow stem cells (BMSCs) were isolated from bone marrow of canine ilium and cultured in Dulbecco's modified eagle medium:nutrient mixture F-12 culture media for 1 week and the second-generation BMSCs were further induced by osteogenic medium (1×10(-8) mol/L dexamethasone, 10 mmol/L B-sodium glycerophosphate and 50 µg/ml vitamin C) and by endothelial cell grow medium (vascular endothelial growth factor and basic fibroblast growth factor) for 14 days in vitro. Thus BMSCs were induced into ECs and OBs. After the second passage, cells were digested and collected.And cell density was adjusted to 1.0×10(6)/ml.The cells and nHA/RHLC/PLA scaffold were co-cultured for 2-4 hours then nHA/RHLC/PLA scaffold composites prepared. Cavity defects of 8 mm in diameter and 10 mm in height were made in femoral heads.The nHA/RHLC/PLA scaffold composited with ECs and osteoblasts (OBs) (group A) and composited with OBs (group B) were inserted into different defects while cell-free nHA/RHLC/PLA scaffold served as controls (group C). New bone formation and defect repair were evaluated at 3 and 6 months by radiographic examination, histology and bone histomorphometry. RESULTS: New bone formation was evident as early as 3 months in groups A, B and C.At 6 months, abundant bone tissue within defects was observed in group A. The control animals with cell-free scaffold showed less bone formation at both timepoints.The scaffold of nHA/RHLC/PLA was degraded and absorbed gradually with the formation of new bone tissues.Histology and bone histomorphometry further revealed significantly increased trabecular bones in group A compared with groups B and C at 6 months postimplantation (P < 0.01). CONCLUSION: More abundant new bone tissue may be found in the bone defect areas implanted with osteoblast-endotheliocyte composite than osteoblasts composite and scaffold materials only.ECs and osteoblasts derived from BMSC are ideal seed cells for repairing femoral head defects.