Inhibition of spastin impairs motor function recovery after spinal cord injury.

Promoting axonal regeneration is an effective strategy for recovery from traumatic spinal cord injury (SCI). Spastin, a microtubule-severing protein, modulates axonal outgrowth and branch formation by regulating microtubule dynamics. However, the exact role of spastin during recovery from SCI remains unknown. Therefore, we utilized a hemisection injury model of the mouse spinal cord and explored the effect of spastin using a spastin inhibitor, spastazoline. Results showed that spastazoline significantly suppressed the microtubule-severing activity of spastin in COS-7 cells and inhibited the promoting effect of spastin on neurite outgrowth in primarily cultured hippocampal neurons. The protein expression level of spastin was significantly upregulated in the injured spinal cord. Injured mice showed impaired motor functions, which included increased toe-off angle and foot fault steps and decreased stride length and Basso mouse scale score. Notably, these motor function impairments were aggravated by the application of spastazoline. Inhibition of spastin exacerbated neurogenesis impairment, as demonstrated by neuronal nuclei antigen staining, the inflammatory response, as shown by Iba-1 and GFAP staining, and axonal regeneration impairment, as shown by 5-hydroxytryptamine staining. Furthermore, mass spectrometry analysis revealed that the inhibition of spastin resulted in numerous dysregulated differentially expressed proteins that were closely associated with vesicle organization and transport. Taken together, our data suggest that spastin is critical for recovery from SCI and may be a potential target for the treatment of SCI.

Phosphorylation mutation impairs the promoting effect of spastin on neurite outgrowth without affecting its microtubule severing ability.

Spastin, a microtubule-severing enzyme, is known to be important for neurite outgrowth. However, the role of spastin post-translational modification, particularly its phosphorylation regulation in neuronal outgrowth, remains unclear. This study aimed to investigate the effects of eliminating spastin phosphorylation on the neurite outgrowth of rat hippocampal neurons. To accomplish this, we constructed a spastin mutant with eleven potential phosphorylation sites mutated to alanine. The phosphorylation levels of the wildtype spastin (WT) and the mutant (11A) were then detected using Phos-tag SDS-PAGE. The spastin constructs were transfected into COS7 cells for the observation of microtubule severing, and into rat hippocampal neurons for the detection of neuronal outgrowth. The results showed that compared to the spastin WT, the phosphorylation levels were significantly reduced in the spastin 11A mutant. The spastin mutant 11A impaired its ability to promote neurite length, branching, and complexity in hippocampal neurons, but did not affect its ability to sever microtubules in COS7 cells. In conclusion, the data suggest that mutations at multiple phosphorylation sites of spastin do not impair its microtubule cleavage ability in COS7 cells, but reduce its ability to promote neurite outgrowth in rat hippocampal neurons.

Development and validation of a nomogram to predict the 30-day mortality risk of patients with intracerebral hemorrhage.

Background: Intracerebral hemorrhage (ICH) is a stroke syndrome with an unfavorable prognosis. Currently, there is no comprehensive clinical indicator for mortality prediction of ICH patients. The purpose of our study was to construct and evaluate a nomogram for predicting the 30-day mortality risk of ICH patients. Methods: ICH patients were extracted from the MIMIC-III database according to the ICD-9 code and randomly divided into training and verification cohorts. The least absolute shrinkage and selection operator (LASSO) method and multivariate logistic regression were applied to determine independent risk factors. These risk factors were used to construct a nomogram model for predicting the 30-day mortality risk of ICH patients. The nomogram was verified by the area under the receiver operating characteristic curve (AUC), integrated discrimination improvement (IDI), net reclassification improvement (NRI), and decision curve analysis (DCA). Results: A total of 890 ICH patients were included in the study. Logistic regression analysis revealed that age (OR = 1.05, P < 0.001), Glasgow Coma Scale score (OR = 0.91, P < 0.001), creatinine (OR = 1.30, P < 0.001), white blood cell count (OR = 1.10, P < 0.001), temperature (OR = 1.73, P < 0.001), glucose (OR = 1.01, P < 0.001), urine output (OR = 1.00, P = 0.020), and bleeding volume (OR = 1.02, P < 0.001) were independent risk factors for 30-day mortality of ICH patients. The calibration curve indicated that the nomogram was well calibrated. When predicting the 30-day mortality risk, the nomogram exhibited good discrimination in the training and validation cohorts (C-index: 0.782 and 0.778, respectively). The AUCs were 0.778, 0.733, and 0.728 for the nomogram, Simplified Acute Physiology Score II (SAPSII), and Oxford Acute Severity of Illness Score (OASIS), respectively, in the validation cohort. The IDI and NRI calculations and DCA analysis revealed that the nomogram model had a greater net benefit than the SAPSII and OASIS scoring systems. Conclusion: This study identified independent risk factors for 30-day mortality of ICH patients and constructed a predictive nomogram model, which may help to improve the prognosis of ICH patients.

[Spatio-temporal variations in species composition and diversity of fish communities in the adjacent waters of Changshan Islands, China]. / é•¿å±±åˆ—å²›é‚»è¿‘æµ·åŸŸé±¼ç±»ç¾¤è½ç§ç±»ç»„æˆå’Œå¤šæ ·æ€§æ—¶ç©ºå˜åŒ–.

In order to understand species composition and diversity of fish communities in the ecotone between the Yellow Sea and Bohai Sea, we examined species composition and temporal and spatial variations of fish species diversity using the relative importance index (IRI), species diversity index and k dominance curve based on bottom trawl survey data of fish resources in the adjacent waters of the Changshan Islands in October 2016, January, May and August 2017. The results showed that 77 fish species were captured, mainly temperate water fishes, demersal fishes and migratory fishes. The dominant species exhibited obvious seasonal variation. The dominant species in spring and winter were Lophius litulon, and those in summer were pelagic species such as Scomber japonicus and Engraulis japonicus. A total of 46 migratory fish species were found throughout the year. The seasonal species migration indices were all higher than 100, while the species migration index was the largest in autumn. The species diversity index showed high spatial and temporal dynamics, with the highest species richness in spring and the highest Shannon diversity and evenness in autumn. There was a significant negative correlation between species richness and sea surface temperature in summer, significant positive correlations between species richness and depth and bottom temperature in winter, and a significant positive correlation between Shannon diversity index and sea bottom temperature. In short, fish community showed high species diversity, rich migratory species and obvious temporal and spatial heterogeneity in the adjacent waters of the Changshan Islands, which is an ecotone between the Yellow Sea and the Bohai Sea.

SUMOylation of microtubule-cleaving enzyme KATNA1 promotes microtubule severing and neurite outgrowth.

Katanin p60 ATPase-containing subunit A1 (KATNA1) is a microtubule-cleaving enzyme that regulates the development of neural protrusions through cytoskeletal rearrangements. However, the mechanism underlying the linkage of the small ubiquitin-like modifier (SUMO) protein to KATNA1 and how this modification regulates the development of neural protrusions is unclear. Here we discovered, using mass spectrometry analysis, that SUMO-conjugating enzyme UBC9, an enzyme necessary for the SUMOylation process, was present in the KATNA1 interactome. Moreover, GST-pull down and co-immunoprecipitation assays confirmed that KATNA1 and SUMO interact. We further demonstrated using immunofluorescence experiments that KATNA1 and the SUMO2 isoform colocalized in hippocampal neurites. We also performed a bioinformatics analysis of KATNA1 protein sequences to identify three potentially conserved SUMOylation sites (K77, K157, and K330) among vertebrates. Mutation of K330, but not K77 or K157, abolished KATNA1-induced microtubule severing and decreased the level of binding observed for KATNA1 and SUMO2. Cotransfection of SUMO2 and wildtype KATNA1 in COS7 cells increased microtubule severing, whereas no effect was observed after cotransfection with the K330R KATNA1 mutant. Furthermore, in cultured hippocampal neurons, overexpression of wildtype KATNA1 significantly promoted neurite outgrowth, whereas the K330R mutant eliminated this effect. Taken together, our results demonstrate that the K330 site in KATNA1 is modified by SUMOylation and SUMOylation of KATNA1 promotes microtubule dynamics and hippocampal neurite outgrowth.

Copper-Catalyzed Aminoarylation of Alkenes via Aminyl Radical Addition and Aryl Migration.

We describe a new strategy for aminoarylation of alkenes by copper-catalyzed smiles rearrangement using O-benzoylhydroxylamines as the amine reagent. This method affords various ß-amino amide derivatives possessing a quaternary carbon center with wide functional group tolerance and high regioselectivity. The mechanistic studies indicate that the transformation can involve aminyl radical intermediates under acid-free condition.

Different fusion tags affect the activity of ubiquitin overexpression on spastin protein stability.

Spastin is one of the proteins which lead to hereditary spastic paraplegia (HSP), whose dysfunction towards microtubule severing and membrane transporting is critically important. The present study is to elucidate the mechanisms of the protein stability regulation of spastin. The ubiquitin encoding plasmids are transfected into COS-7 cells with different fusion tags including Green Fluorescent Protein (GFP), mCherry and Flag. The expression level of spastin was detected, microtubule severing activity and neurite outgrowth were quantified. The data showed that ubiquitin overexpression significantly induced the decreased expression of spastin, suppressed the activity of microtubule severing in COS-7 cells and inhibited the promoting effect on neurite outgrowth in cultured hippocampal neurons. Furthermore, when modulating the overexpression experiments of ubiquitin, it was found that relatively small tag like Flag, but not large tags such as GFP or mCherry fused with ubiquitin, retained the activity on spastin stability. The present study investigated the effects of small/large tags addition to ubiquitin and the novel mechanisms of post-transcriptional modifications of spastin on regulating neurite outgrowth, in the attempt to experimentally elucidate the mechanisms that control the level or stability of spastin in hereditary spastic paraplegia.

Photoredox-catalyzed redox-neutral difluoroalkylation to construct perfluoroketones with difluoroenoxysilanes.

A mild and facile approach to construct various perfluoroketones via photo-catalyzed difluoroalkylation of difluoroenoxysilanes is developed. The reaction includes a strategy of combination of two fluorine-containing functional groups, which confers the reaction with characteristics like high efficiency, mild conditions, and broad scope. A variety of fluoroalkyl halides including perfluoroalkyl iodides, bromo difluoro esters and amides can be employed as radical precursors. Control experiments indicate that a single-electron transfer pathway may be involved in the reaction.

Investigation of normal knees kinematics in walking and running at different speeds using a portable motion analysis system.

Walking and running at different speeds are common in daily life. This study investigated 6 degrees of freedom (DOF) kinematics of normal knees of Chinese during walking and running. Forty healthy participants were investigated in 4 conditions: comfortable walking, normal walking, slow running and ordinary running. The range of motion (ROM) and peak values in 6 DOF kinematics were analysed. As the speed increased, a general increase in flexion, lateral and proximal translations occurred. Significant increases of ROM in flexion/extension, axial rotation and medial/lateral translations were observed. The ROM of adduction/abduction, anterior/posterior and proximal/distal translations were greatest during normal walking. The maximum and minimum flexion/extension, maximum internal rotation and tibial lateral translations increased with the increase of speed. The maximum and minimum tibial proximal translations in running were found being greater than walking. A phenomenon between walking and running was observed: both tibial proximal/distal and medial/lateral translations increased when changed from walking to running. Non-linear transition exists in 6 DOF kinematics during walking to running. Discoveries in this study may have potential clinical values to serve as references of normal walking and running in the management of knee injury and knee rehabilitation.

The lncRNA MALAT1/miR-30/Spastin Axis Regulates Hippocampal Neurite Outgrowth.

Spastin, a microtubule-severing enzyme, is important for neurite outgrowth. However, the mechanisms underlying the post-transcriptional regulation of spastin during microtubule-related processes are largely unknown. We demonstrated that the spastin expression level is controlled by a long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-30 (miR-30) axis during neurite outgrowth. The miR-30 expression level decreased in hippocampal neurons with increasing days in culture, and miR-30 overexpression suppressed while miR-30 inhibition promoted neurite outgrowth in hippocampal neurons. Spastin was validated as a target gene of miR-30 using the luciferase reporter assay. The protein expression, microtubule severing activity, and neurite promoting effect of spastin were suppressed by the overexpression of miR-30 mimics and increased by miR-30 inhibitors. MALAT1 expression increased during neurite outgrowth and MALAT1 silencing impaired neurite outgrowth. miR-30 was a sponge target of MALAT1 and MALAT1/miR-30 altered neurite outgrowth in hippocampal neurons. MALAT1 overexpression reversed the inhibitory effect of miR-30 on the activity of a luciferase reporter construct containing spastin, as well as spastin mRNA and protein expression, indicating that spastin was a downstream effector of MALAT1/miR-30. The MALAT1/miR-30 cascade also modulated spastin-induced microtubule severing, and the MALAT1/miR-30/spastin axis regulated neurite outgrowth in hippocampal neurons. This study suggests a new mechanism governing neurite outgrowth in hippocampal neurons involving MALAT1/miR-30-regulated spastin expression.

Progress of co-culture systems in cartilage regeneration.

INTRODUCTION: Cartilage tissue engineering has rapidly developed in recent decades, exhibiting promising potential to regenerate and repair cartilage. However, the origin of a large amount of a suitable seed cell source is the major bottleneck for the further clinical application of cartilage tissue engineering. The use of a monoculture of passaged chondrocytes or mesenchymal stem cells results in undesired outcomes, such as fibrocartilage formation and hypertrophy. In the last two decades, co-cultures of chondrocytes and a variety of mesenchymal stem cells have been intensively investigated in vitro and in vivo, shedding light on the perspective of co-culture in cartilage tissue engineering. AREAS COVERED: We summarize the recent literature on the application of heterologous cell co-culture systems in cartilage tissue engineering and compare the differences between direct and indirect co-culture systems as well as discuss the underlying mechanisms. EXPERT OPINION: Co-culture system is proven to address many issues encountered by monocultures in cartilage tissue engineering, including reducing the number of chondrocytes needed and alleviating the dedifferentiation of chondrocytes. With the further development and knowledge of biomaterials, cartilage tissue engineering that combines the co-culture system and advanced biomaterials is expected to solve the difficult problem regarding the regeneration of functional cartilage.