Comparative proteomes change and possible role in different pathways of microRNA-21a-5p in a mouse model of spinal cord injury.

Our previous study found that microRNA-21a-5p (miR-21a-5p) knockdown could improve the recovery of motor function after spinal cord injury in a mouse model, but the precise molecular mechanism remains poorly understood. In this study, a modified Allen's weight drop was used to establish a mouse model of spinal cord injury. A proteomics approach was used to understand the role of differential protein expression with miR-21a-5p knockdown, using a mouse model of spinal cord injury without gene knockout as a negative control group. We found that after introducing miR-21a-5p knockdown, proteins that played an essential role in the regulation of inflammatory processes, cell protection against oxidative stress, cell redox homeostasis, and cell maintenance were upregulated compared with the negative control group. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis identified enriched pathways in both groups, such as the oxidative phosphorylation pathway, which is relevant to Parkinson's disease, Huntington's disease, Alzheimer's disease, and cardiac muscle contraction. We also found that miR-21a-5p could be a potential biomarker for amyotrophic lateral sclerosis, as miR-21a-5p becomes deregulated in this pathway. These results indicate successful detection of some important proteins that play potential roles in spinal cord injury. Elucidating the relationship between these proteins and the recovery of spinal cord injury will provide a reference for future research of spinal cord injury biomarkers. All experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of Shandong University of China on March 5, 2014.

Neuroprotective mechanisms of rutin for spinal cord injury through anti-oxidation and anti-inflammation and inhibition of p38 mitogen activated protein kinase pathway.

Rutin has anti-inflammatory, antioxidant, anti-viral, anti-tumor and immune regulatory effects. However, the neuroprotective effects of rutin in spinal cord injury are unknown. The p38 mitogen activated protein kinase (p38 MAPK) pathway is the most important member of the MAPK family that controls inflammation. We assumed that the mechanism of rutin in the repair of spinal cord injury is associated with the inhibition of p38 MAPK pathway. Allen's method was used to establish a rat model of spinal cord injury. The rat model was intraperitoneally injected with rutin (30 mg/kg) for 3 days. After treatment with rutin, Basso, Beattie and Bresnahan locomotor function scores increased. Water content, tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 levels, p38 MAPK protein expression and caspase-3 and -9 activities in T8-9 spinal cord decreased. Oxidative stress related markers superoxide dismutase and glutathione peroxidase levels increased in peripheral blood. Rutin exerts neuroprotective effect through anti-oxidation, anti-inflammation, anti-apoptosis and inhibition of p38 MAPK pathway.

Regulatory roles of microRNA-21 in fibrosis through interaction with diverse pathways (Review).

MicroRNA-21 (miR-21) is a small, non-coding RNA which can regulate gene expression at the post­transcriptional level. While the fibrogenic process is vital in tissue repair, proliferation and transition of fibrogenic cells combined with an imbalance of secretion and degradation of the extracellular matrix results in excessive tissue remodeling and fibrosis. Recent studies have indicated that miR­21 is overexpressed during fibrosis and can regulate the fibrogenic process in a variety of organs and tissues via diverse pathways. The present review summarized the significant roles of miR-21 in fibrosis and discussed the underlying key pathways.

[The changes of extracellular matrix in adult degenerative nucleus pulposus cells with stiring microcarrier culture system in vitro].

OBJECTIVE: To evaluate the biological effect on the synthesis of the extracellular matrix (ECM) in the cultivation of adult degenerative nucleus pulposus cells using the stiring microcarrier system in vitro. METHODS: Thirty-four specimens were collected after intervertebral fusion operations of the patients with intervertebral disc herniation diseases from September 2005 to May 2009. The specimens were then randomly allocated into 2 groups for in vitro cultivation: monolayer culture group and microcarrier culture group. On the exponential phase, SP-ABC immunohistochemical staining and Western blot quantitative analysis were conducted in the two groups to detect the collagen type I and II. Proteoglycan contents of two groups in different growth phases were detected with (35)S-sulfate incorporation assay. RESULT: The expressions of collagen type I and II in microcarrier culture group were significantly higher than those in monolayer culture group: SP-ABC immunohistochemical staining (collagen type I: 32.5 ± 4.4 vs. 15.2 ± 1.2, t = 2.871, P < 0.01; collagen type II: 43.6 ± 4.1 vs. 23.1 ± 2.2, t = 2.375, P < 0.05); Western blot quantitative analysis (collagen type I: 0.62 ± 0.08 vs. 0.50 ± 0.06, t = 3.327, P < 0.01; collagen type II: 1.46 ± 0.08 vs. 0.86 ± 0.04, t = 2.453, P < 0.05). Nucleus pulposus cells cultivated in stiring microcarrier system showed significantly increased proteoglycan synthesis than monolayer culture group does on both exponential phase and stationary phase (exponential phase: 34 821 ± 312 vs. 21 046 ± 673, t = 2.134, P < 0.05; stationary phase: 45 134 ± 175 vs. 32 193 ± 713, t = 2.801, P < 0.01). CONCLUSIONS: The expression of collagen type I, II and proteoglycan of adult degenerative nucleus pulposus cells are positive regulated by the stiring microcarrier system, which can be used in the mass amplification of the adult degenerative nucleus pulposus cells.

Variation in sexual behaviors in a group of captive male Yangtze finless porpoises (Neophocaena phocaenoides asiaeorientalis): motivated by physiological changes?

Most male mammals in temperate regions demonstrate seasonal sexual behaviors that coincide with seasonal variations in gonadal activities and androgen hormones. The Yangtze finless porpoise is a temperate freshwater cetacean species and an obvious seasonal breeder. To investigate the relationship between sexual behavior and gonadal activity in this animal, testicular size (volume) and structure (ultrasonogram pixel intensity) of two adult male porpoises (AF, AB) and one sub-adult male (TT) were longitudinally monitored from November 2008 to November 2009. Serum testosterone concentration was also monitored during the same period. Variations in the frequency of sexual behavior in AF and AB had similar, but seasonal trends. Their testicular size and pixel intensity also varied seasonally. Testicular size increased in March, peaked from April through June, and decreased gradually from August through September, whereas testicular pixel intensity started to increase in early February. The frequency of sexual behavior was positively correlated with testicular volume and pixel intensity (P = 0.000018 and P = 0.00012, respectively) in AF. Serum testosterone concentrations also varied. The sub-adult male porpoise, TT, was undergoing puberty, as evidenced by its marked increase in testicular volume, testicular pixel intensity, and serum testosterone concentrations from the beginning of 2009. Interestingly, TT exhibited the highest frequency of sexual behavior, most of which was same-sex pairing. However, its oversexed behavior neither quantitatively correlated with its smaller testicular volume (P = 0.61) nor with its testicular pixel intensity (P = 0.69).

Circulating blood monocytes traffic to and participate in the periprosthetic tissue inflammation.

OBJECTIVE: To examine the trafficking of human circulating blood monocytes and their influence on the inflammation of periprosthetic tissues using a novel mouse-human chimera model. METHODS: Periprosthetic tissue and bone chips from patients with aseptic prosthetic loosening were implanted into the muscles of immune-deficient SCID mice depleted of host macrophages by periodic intraperitoneal injection of anti-asialo GM1 rabbit sera (ASGM1). Autologous patient peripheral blood monocytes (PBMCs) were labeled with PKH2 fluorescent dye and injected intraperitoneally into the implanted animals. Mice were sacrificed 14 days after PBMC transfusion for molecular and histological analyses. RESULTS: Patient periprosthetic tissues were well tolerated in SCID mice and preserved a high level of viability. Cell trafficking studies revealed the accumulation of fluorescent PBMC within the xenografts, with total cell counts in the xenografts significantly increased following the systemic PBMC infusion. PBMC infusion also promoted the expression of IL-1, IL-6, TNFalpha, and RANK within the periprosthetic tissue. CONCLUSION: Systemic PBMC migrated to the implanted periprosthetic tissues and contributed to the local inflammation. The data provide evidence that circulating blood monocytes may play a role in pathologic process during aseptic loosening of total joint replacement.

Destructive pathological changes in the rat spinal cord due to chronic mechanical compression. Laboratory investigation.

OBJECT: Chronic mechanical compression of the spinal cord, which is commonly caused by degeneration of the spine, impairs motor and sensory functions insidiously and progressively. Yet the exact mechanisms of chronic spinal cord compression (SCC) remain to be elucidated. To study the pathophysiology of this condition, the authors developed a simple animal experimental model that reproduced the clinical course of mechanical compression of the spinal cord. METHODS: A custom-designed compression device was implanted on the exposed spinal cord of female Wistar rats between the T-7 and T-9 vertebrae. A root canal screw attached to a plastic plate was tightened 1 complete turn (1 pitch) every 7 days for 6 weeks. The placement of the compression device and the degree of compression were validated every week using radiography. Furthermore, a motor sensory deficit index was also calculated every week. After 3, 6, 9, or 12 weeks, the compressed T7-9 spinal cords were harvested and examined histologically. RESULTS: Lateral projection of the thoracic spine showed a progressively increasing rate of mean spinal cord narrowing in the compression group. Motor and sensory deficiencies were observed from Week 3 onward; paralysis was observed in 2 rats at Week 12. Motor deficiency appeared earlier than sensory deficiency. Obvious pathological changes were observed starting at Week 6. The number of neurons in the gray matter of rats with chronic compression of the spinal cord decreased progressively in the 6- and 9-week compression groups. In the white matter, myelin destruction and loss of axons and glia were noted. The number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive neurons increased in the ventral-to-dorsal direction. The number of TUNEL-positive cells increased from Week 6 onward and peaked at Week 9. CONCLUSIONS: This practical model accurately reproduces characteristic features of clinical chronic SCC, including progressive motor and sensory disturbances after a latency and insidious neuronal loss.

Purification, crystallization and preliminary X-ray studies of a p-nitrophenylphosphatase from Bacillus stearothermophilus.

Thermostable p-nitrophenylphosphatase from Bacillus stearothermophilus has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group C(2), with unit-cell parameters a = 67.17 A, b = 57.84 A, c = 62.49 A and alpha = 90.0 degrees, beta = 95.4 degrees, gamma = 90.0 degrees. Diffraction data were collected to 1.40 A resolution with a completeness of 94.7% (96.6% for the last shell), an R(fac) value of 0.074 (0.341) and an I/sigma (I) value of 30.1 (2.67).