[Comparison of the outcomes between anterior cervical discectomy and fusion versus posterior laminectomy and fusion for the treatment of multi-level cervical spondylotic myelopathy combined with cervical kyphosis].

Objective: To compare the outcomes between anterior cervical discectomy and fusion (ACDF) and posterior laminectomy and fusion(LF) for multilevel cervical spondylotic myelopathy combined with cervical kyphosis. Methods: From January 2010 to June 2014, 54 patients with cervical spondylotic myelopathy combined with cervical kyphosis underwent surgical treatment.Among them, 29 patients were underwent ACDF, and 25 patients were underwent LF in Department of spine surgery, Tianjin Union Medical Centre. The operation time, intraoperative blood loss, fusion segments, Japanese Orthopaedic Association (JOA)score, Neck Disability Index (NDI), Visual Analog Scale (VAS), change of cervical curvature, range of motion(ROM)and complications were recorded and compared between the two groups. Results: Mean operative time was (162.7±21.3)min in the anterior approach group versus (176.3±29.8)min in the posterior group(P>0.05). Mean intraoperative blood loss was (135.6±27.8)ml in the anterior approach group and (255.2±32.3)ml in the posterior approach group(P<0.05). Mean fusion levels are (4.1±0.3)in the anterior approach group and (5.3±0.5) in the posterior approach group(P<0.05). The mean preoperative JOA score were(8.3±2.7)in the anterior approach group and( 8.9±2.1) in the posterior approach group (P>0.05). Mean postoperative JOA score were(13.6±2.5) in the anterior approach group and (14.0±1.7)in the posterior approach group at final follow-up(P>0.05). Mean improvement rate was (55.7%±16.3%)in the anterior approach group and (58.3%±15.7%) in the posterior approach group (P>0.05). Mean preoperative NDI score were(33.8±11.0)in the anterior approach group and (34.4±8.7)in the posterior approach group (P>0.05). Mean postoperative NDI score were (16.9±7.5) in the anterior approach group and (15.5±8.1) in the posterior approach group at final follow-up (P>0.05). Mean VAS score were (2.9±1.5) in the anterior approach group and (2.5±1.0) in the posterior approach group before operation(P>0.05), they are improved to (1.2±1.2) and (1.2±1.3), respectively(P>0.05). Mean Cobb angle of the operative site were (-24.3±4.4)°in the anterior approach group and (-22.7±3.7)° in the posterior approach group before operation(P>0.05). At final follow-up, the Cobb angle of the operative site were (13.7±3.2)°in the anterior approach group and (6.2±4.2)° in the posterior approach group(P<0.01). Mean preoperative ROM were (29.0±6.7)°and (30.4±5.4)° in the anterior approach group and posterior approach group, respectively(P>0.05). Mean postoperative ROM were (11.7±6.5)° and (8.2±5.9)°in the anterior approach group and the posterior approach group, respectively(P<0.05). There were 16 patients with complications in the anterior approach group and 7 patients with complications in the posterior approach group(P<0.05). Conclusion: For multilevel cervical spondylotic myelopathy combined with cervical kyphosis, ACDF can restore the lordosis better, fuse less levels but have more complications compared with LF. Patients treated with LF can get as good life quality as with ACDF and have less complications although fuse more levels compared with ACDF.

Correlation and the mechanism of lithium ion diffusion with the crystal structure of Li7La3Zr2O12 revealed by an internal friction technique.

The correlation and transport mechanism of lithium ions with the crystal structure of a fast lithium ion conductor Li7La3Zr2O12 are mainly investigated by internal friction (IF) and AC impedance spectroscopy techniques. Compared with the poor conductivity of tetragonal Li7La3Zr2O12, the Al stabilized cubic phase exhibits a good ionic conductivity that can be up to 1.9 × 10(-4) S cm(-1) at room temperature, which can be ascribed to the disordered distribution of lithium ions in the cubic phase. A well-pronounced relaxation IF peak (labeled as peak PC) is observed in the cubic phase while a very weak IF peak (labeled as PT) is observed in the tetragonal phase, further evidencing the difference in lithium ion migration in the two phases. Peak PC can be decomposed into two sub-peaks with the activation energy and the pre-exponential factor of relaxation time being E1 = 0.41 eV and τ01 = 1.2 × 10(-14) s for the lower temperature peak PC1 and E2 = 0.35 eV and τ02 = 1.9 × 10(-15) s for the higher temperature PC2 peak, respectively. Based on the crystalline structure of a cubic garnet-type Li7La3Zr2O12 compound, an atomistic mechanism of lithium ion diffusion via vacancies is suggested, i.e. 48g(96h) ↔ 48g(96h) for peak PC1 and 48g(96h) ↔ 24d for peak PC2, respectively. The weak PT peak in the tetragonal phase is preliminarily interpreted as due to the short jump process among neighboring octahedral sites and vacant tetrahedral sites.

In vivo mechanism study of NGAL in rat renal ischemia-reperfusion injury.

This study aimed to determine the protective effect and mechanism of neutrophil gelatinase-associated lipocalin (NGAL) in rat kidney on ischemia/reperfusion injury (I/R). The rat I/R model was set up by cutting one kidney and clamping the contralateral renal pedicle for 45 min. Male SD rats were randomly divided into sham-operation, I/R and NGAL groups. Hematoxylin-eosin staining was performed to observe the renal pathological changes in the 3 groups; serum creatinine (Scr) and blood urea nitrogen (BUN) determined in blood samples taken from the inferior vena cava 24 h after the reperfusion were measured; TUNEL was used to observe the apoptosis of renal tubular epithelial cells; immunohistochemistry was performed to evaluate the expressions of Bax and activated caspase-3; Western blotting was used to determine the expression changes in apoptotic proteins Fas and Bcl-2. Compared with the I/R group, Scr and BUN of the NGAL group were 63.400 ± 11.908 vs 121.857 ± 17.151 µM and 14.840 ± 2.868 vs 28.557 ± 6.434 mM, respectively. The number of apoptotic tubular epithelial cells was reduced (7.800 ± 1.924 vs 15.400 ± 3.049); the expression of renal tissue Fas mRNA of the NGAL group was decreased (2.34 ± 0.51 vs 6.84 ± 2.34); the expression of the Bax protein was lower (7.440 ± 1.640 vs 15.456 ± 1.955%); the expression of the CC3 protein was decreased (3.171 ± 0.321 vs 7.291 ± 1.059%), while the expression of the Bcl-2 protein increased (6.91 ± 1.64 vs 5.30 ± 1.48), P < 0.05. NGAL had a protective effect towards the renal tubular epithelial cells in I/R, and the effect might have been associated with the reduction in apoptosis and the altered expression of apoptotic proteins, which would thereby reduce tissue damage and protect the kidney.

Differential activation of migration by hypoxia in keratinocytes isolated from donors of increasing age: implication for chronic wounds in the elderly.

Chronic wound healing conditions are often observed in elderly patients with poor tissue oxygenation. Impaired re-epithelialization is a hallmark of these wounds, which is seen in both clinical studies and in our animal models of impaired healing. To investigate the pathogenic mechanism of chronic wounds, we studied the effect of hypoxia on migration of keratinocytes isolated from human donors of increasing age. Keratinocytes from elderly donors had depressed migratory activity when exposed to hypoxia, as opposed to an increase in migration in young cells. Analysis of underlying biochemical changes demonstrated a differential activation of matrix metalloproteinases by hypoxia in keratinocytes isolated from the young and the old. Matrix metalloproteinases-1 and -9 and tissue inhibitor of matrix metalloproteinase-1 were strongly upregulated by hypoxia in young cells, whereas no induction was observed in aged cells. Furthermore, transforming growth factor-beta 1 signaling appears to be involved in the keratinocyte differential response to hypoxia, as transforming growth factor-beta type I receptor was upregulated by hypoxia in young cells, while there was no induction in aged cells. Transforming growth factor-beta neutralizing reagents blocked hypoxia-induced matrix metalloproteinase-1, matrix metalloproteinase-9 expression, and hypoxia-induced cell migration as well. Our results suggest that an age-related decrease in response to hypoxia plays a crucial part in the pathogenesis of retarded re-epithelialization in wound.

Solubility of phosphorus and heavy metals in potting media amended with yard waste-biosolids compost.

The potential risk of surface and ground water contamination by phosphorus (P) and heavy metals leached from compost-based containerized media has become an environmental concern. Solubility and fractionation of P and heavy metals were evaluated in media containing 0, 25, 50, 75, or 100% compost derived from biosolids and yard trimmings for potential impacts on the environment. As compost proportion in peat-based media increased from 0 to 100%, concentrations of total P, Cd, Cu, Ni, Pb, Zn, and Mn in the media increased whereas concentrations of total Co and Cr decreased. Except for Cu, all heavy metals in the water-soluble fraction decreased with increasing compost proportion in the media, because of higher Fe, Al, and Ca concentrations and pH values of the composts than the peat. When the media pH is controlled and maintained at normal range of plant growth (5.5-6.5), leaching of the heavy metals is minimal. Incorporation of compost to the peat-based media also decreased the proportion of total P that was water-soluble. However, concentrations of bioavailable inorganic phosphorus (NaHCO3-IP), readily mineralizable organic phosphorus (NaHCO3-OP), potentially bioavailable inorganic phosphorus (NaOH-IP), and potentially bioavailable organic phosphorus (NaOH-OP) were still higher in the media amended with compost because of higher total P concentration in the compost. Further study is needed to verify if less or no topdressing of chemical P fertilizer should be applied to the compost-amended media to minimize P effect on the environment when compost-amended potting media are used for nursery or greenhouse crop production systems.

Exendin-4 improved rat cortical neuron survival under oxygen/glucose deprivation through PKA pathway.

Previous studies demonstrated that exendin-4 (Ex-4) may possess neurotrophic and neuroprotective functions in ischemia insults, but its mechanism remained unknown. Here, by using real-time PCR and ELISA, we identified the distribution of active GLP-1Rs in the rat primary cortical neurons. After establishment of an in vitro ischemia model by oxygen/glucose deprivation (OGD), neurons were treated with various dosages of Ex-4. The MTT assay showed that the relative survival rate increased with the dosage of Ex-4 ranging from 0.2 to 0.8 µg/ml (P<0.001, vs. OGD group). The apoptosis rate was reduced from (49.47±2.70)% to (14.61±0.81)% after Ex-4 treatment (0.4 µg/ml) 12h after OGD (P<0.001). Moreover, immunofluorescence staining indicated that Ex-4 increased glucose-regulated proteins 78 (GRP78) and reduced C/EBP-homologous protein (CHOP). Western blot analysis demonstrated that, after neurons were treated with Ex-4, GRP78 was up-regulated over time (P<0.01, vs. OGD group), while CHOP levels rose to a peak 8h after OGD and then decreased (P<0.05, vs. OGD group). This effect was changed by both the protein kinase A (PKA) inhibitor H89 (P<0.01, P<0.05, respectively, vs. Ex-4 group) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (P<0.01, P<0.01, respectively, vs. Ex-4 group) but not by the mitogen-activated protein kinase (MAPK) inhibitor U0126. Our study also revealed that, compared with the Ex-4 group, inhibition of the PKA signaling pathway significantly decreased the survival rate of neurons, down-regulated the expression of B-cell lymphoma 2 (Bcl-2) and up-regulated the Bax expression 3h after ODG (P<0.05, P<0.01, respectively), while neither PI3K nor MAPK inhibition exerted such effects. Furthermore, Western blotting exhibited that PKA expression was elevated in the presence or absence of OGD insults (P<0.05). This study indicated that Ex-4 protected neurons against OGD by modulating the unfolded protein response (UPR) through the PKA pathway and may serve as a novel therapeutic agent for stroke.

The protective effect of sonic hedgehog is mediated by the phosphoinositide [corrected] 3-kinase/AKT/Bcl-2 pathway in cultured rat astrocytes under oxidative stress.

In our previous study, we found that the sonic hedgehog (Shh) signaling pathway is activated in neurons under oxidative stress and plays a neuro-protective role [Dai RL, et al. (2011) Neurochem Res 36:67-75]; we are led to postulate that the Shh might be released by astrocytes, thereby protecting neurons against oxidant injury. In primary cultured astrocytes of rats, we found that treatment with 100 µM H2O2 for 24 h induced a significant increase in the mRNA and protein levels of Shh, Patched1, and Gli-1, and the increase was substantially greater in astrocytes than in neurons. In the coculture systems of astrocytes and neurons under the H2O2 treatment, blocking the Shh signaling pathway with 5E1 (an antibody against the N-terminal domain of Shh) could block the neuroprotective activity of astrocytes on cocultured neurons. In this study, we found that treatment with H2O2 (100-800 µM) for 24 h caused cell death of astrocytes in a concentration-dependent manner. MTT reduction and Trypan Blue exclusion assay showed that exogenous Shh increased survival rate of the H2O2-treated astrocytes, whereas pretreatment with cyclopamine (a specific inhibitor of the Shh signaling pathway) or 5E1 decreased the survival rate of the H2O2-treated astrocytes. Shh also inhibited H2O2-induced apoptosis of astrocytes, and this effect could be partially reversed by cyclopamine. We also found that Shh promoted the phosphorylation of AKT, but had no significant effect on p38 or extracellular signal regulated kinases 1 and 2 (ERK 1/2) in H2O2-treated astrocytes. Blocking Shh or phosphoinositide 3-kinases (PI3-K)/AKT signaling pathway with cyclopamine or LY294002 decreased the survival rate of astrocytes, induced cell apoptosis, upregulated the expression of Bax, and downregulated the expression of Bcl-2. We are led to conclude that the oxidative stress induces astrocytes to secrete endogenous Shh and exogenous administration of Shh might protect the astrocytes from oxidative stress by activating PI3-K/AKT/Bcl-2 pathway.

Oligomerization of hepatitis delta antigen is required for both the trans-activating and trans-dominant inhibitory activities of the delta antigen.

Two forms of hepatitis delta antigen (HDAg) have different roles in the replication cycle of hepatitis delta virus (HDV); the small forms trans activates HDV RNA replication, whereas the large form suppresses it but is needed for virion assembly. To understand the mechanism of these regulatory activities, we studied the possible HDAg oligomerization and its role in HDV replication. In this report, we provide direct biochemical evidence for the in vitro and in vivo formation of homodimers and heterodimers between these two HDAg species. By deletion mutagenesis, we showed that this protein interaction is mediated by the leucine zipper-like sequence residing in the N-terminal one-third of HDAg. Furthermore, site-specific mutants with various substitutions on two of the leucine residues in this stretch of sequence had reduced or no ability to form HDAg dimers. Correspondingly, the small HDAg with mutations in the leucine zipper-like sequence had reduced abilities to trans activate HDV RNA replication. Similar mutations on the leucine zipper-like sequence of the large HDAg also resulted in loss of the ability of large HDAg to inhibit HDV RNA replication. The in vivo biological activities of both forms of HDAg (trans activation and trans-dominant inhibition of HDV RNA replication, respectively) correlated with the extent of HDAg oligomerization in vitro. Thus, we conclude that the small HDAg participates in HDV RNA replication as an oligomer form and that the large HDAg inhibits HDV RNA replication as a result of its complex formation with small HDAg. A "black sheep" model for the mechanism of trans-dominant inhibition by the large HDAg is presented.

Heterogeneity of hepatitis delta antigen.

Hepatitis delta antigen (HDAg) is the only known protein encoded by the hepatitis delta virus (HDV). Two HDAg species of different sizes have been detected in the sera and livers of the infected humans, chimpanzees, and woodchucks, even though only one RNA species was previously identified in most of the HDV strains. To study HDAg heterogeneity, we took advantage of the fact that a single base mutation at nucleotide 1015 (C to U), which results in an amber termination codon in the HDAg open reading frame (ORF), eliminates a unique Ncol restriction enzyme site. We screened various HDV cDNA clones and detected sequence heterogeneity of the HDAg-coding region on the basis of the presence or absence of the Ncol site. Five delta hepatitis patients were examined. In every patient, two types of HDAg-coding sequence were detected at nucleotide 1015: one which contains a C and results in an ORF encoding a delta antigen of 214 amino acids, and the other which possesses a U and results in an amber termination codon and a truncated HDAg species of 195 amino acids. The in vitro translation products of these two ORFs comigrated with the two HDAg species from the patient's plasma on SDS polyacrylamide gels. Polymerase chain reaction (PCR) amplification of the HDV RNA from some patients' sera and subsequent sequencing showed several additional mutations in the HDAg-coding region. These mutations are independent of the C or U nucleotide change at the site of the amber termination codon.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta1 fails to stimulate wound healing and impairs its signal transduction in an aged ischemic ulcer model: importance of oxygen and age.

Clinical trials of exogenous growth factors in treating chronic wounds have been less successful than expected. One possible explanation is that most studies used animal models of acute wounds in young animals, whereas most chronic wounds occur in elderly patients with tissue ischemia. We described an animal model of age- and ischemia-impaired wound healing and analyzed the wound-healing response as well as the transforming growth factor (TGF)-beta1 effect in this model. Rabbits of increasing ages were made ischemic in the ear where dermal ulcers were created. Histological analysis showed that epithelium ingrowth and granulation tissue deposition were significantly impaired with increased age under ischemia. TGF-beta1 stimulated wound repair under both ischemic and non-ischemic conditions in young animals, although it showed no statistical difference in aged animals. Procollagen mRNA expression decreased under ischemic conditions and with aging. Neither TGF-beta1 nor procollagen alpha1(I) mRNA expression increased in response to TGF-beta1 treatment under ischemia in aged animals. Therefore, the wound-healing process is impaired additively by aging and ischemia. The lack of a wound-healing response to TGF-beta1 in aged ischemic wounds may play a role in the chronic wounds.

Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex.

Hepatitis delta antigen (HDAg) is the only protein encoded by hepatitis delta virus (HDV). HDAg has been demonstrated in the nuclei of HDV-infected hepatocytes, and its nuclear transport may be important for the replication of HDV RNA. In this report, we investigated the mechanism of nuclear transport of HDAg. By expressing fusion proteins consisting of the different portions of HDAg and alpha-globin, we have identified a nuclear localization signal (NLS) within the N-terminal one-third of HDAg. It consists of two stretches of basic amino acid domains separated by a short run of nonbasic amino acids. Both of the basic domains are necessary for the efficient nuclear transport of HDAg. The nonbasic spacer amino acids could be removed without affecting the nuclear targeting of HDAg significantly. Thus, the HDAg NLS belongs to a newly identified class of NLS which consists of two discontiguous stretches of basic amino acids. This NLS is separated from a stretch of steroid receptor NLS-like sequence, which is also present but not functioning as an NLS, in HDAg. Furthermore, we have shown that subfragments of HDAg which do not contain the NLS can be passively transported into the nucleus by a trans-acting full-length HDAg, provided that these subfragments contain the region with a leucine zipper sequence. Thus, our results indicate that HDAg forms aggregates in the cytoplasm and that the HDAg oligomerization is probably mediated by the leucine zipper sequence. Therefore, HDAg is likely transported into the nucleus as a protein complex.

Expression of the hepatitis delta virus large and small antigens in transgenic mice.

Simultaneous infection with hepatitis delta virus (HDV) and hepatitis B virus (HBV) in humans is often associated with severe viral liver disease including fulminant hepatitis. Since HBV is thought to be noncytopathic to the hepatocyte, the enhanced disease severity observed during dual infection has been attributed to either simultaneous immune responses against the two viruses or direct cytotoxic effects of HDV products on the hepatocyte or both. To examine these alternate possibilities, we produced transgenic mice that express the small and large delta antigens (HDAg) in hepatocyte nuclei at levels equal to those observed during natural HDV infection. No biological or histopathological evidence of liver disease was detectable during 18 months of observation, suggesting that neither the large nor small form of HDAg is directly cytopathic to the hepatocyte in vivo.

Effects of keratinocyte growth factor-2 (KGF-2) on wound healing in an ischaemia-impaired rabbit ear model and on scar formation.

Keratinocyte growth factor-2 (KGF-2), also described as fibroblast growth factor-10 (FGF-10), is a member of the fibroblast growth factor family. KGF-2 shares 57 per cent sequence homology to previously reported KGF-1 (FGF-7). In skin, both growth factors are expressed in the dermal compartment. KGF-1 and KGF-2 bind to the same receptor with high affinity, the KGFR isoform of FGFR2, which is exclusively expressed by epithelial cells. This study examines the in vivo function of topically applied KGF-2 on wound healing using an ischaemia-impaired rabbit dermal ulcer model, in young and aged animals. Histological analysis of the wounds showed that KGF-2 significantly promoted re-epithelialization in both young and old animals. Similar results have been observed with KGF-1 in this model. In addition, KGF-2 enhanced granulation tissue formation in both young and old rabbits, a biological effect not found with KGF-1, suggesting a possible indirect mechanism which enhances neo-granulation tissue formation. Immunohistological staining of day 7 wounds with proliferating cell nuclear antigen (PCNA) antibody demonstrated a significant increase of dermal cell proliferation in KGF-2-treated wounds compared with placebo wounds. These results suggest a mesenchymal-epithelial interaction that is mediated by a paracrine feedback loop of KGF-2. Because of the wound healing impairment observed with ageing, the wound healing response to KGF-2 was also studied in ischaemic wounds of aged animals. Administration of KGF-2 led to significant stimulation of epithelial growth and granulation tissue formation. The effects seen in the old animals were delayed compared with the young animals. Lastly, the effect of KGF-2 was examined in a rabbit model of scar formation. Quantification of scar elevation index showed no significant differences in scar formation when KGF-2 was compared with buffer placebo. Compared with other growth factors, including KGF-1 and TGF-beta which have previously been examined in these models, KGF-2 is the most effective and causes no obvious scarring.