The role of autophagy-related proteins in the pathogenesis of neuromyelitis optica spectrum disorders.

This study aimed to investigate the expression of autophagy-related proteins in a mouse model of neuromyelitis optica (NMO). Mice were assigned to one of four groups: an animal experimental model group (NMO-EAE group, given with exogenous IL-17A), Interleukin-17 monoclonal antibody intervention group (NMO-EAE_0IL17inb), No exogenous interleukin-17 enhanced immune intervention group (NMO-EAE_0IL17), and a control group. Behavioral scores were assessed in each group, and the protein expressions of sequestosome 1 (P62), Beclin-1, the mammalian target of rapamycin (mTOR), phosphoinositide 3-kinase (PI3K-I), and LC3II/LC3I were detected using Western blotting. In the NMO-EAE_0IL17 group, the expression of Beclin-1 decreased, the LC3II/LC3I ratio was lower, and the expressions of P62, mTOR, and PI3K-I increased; after administration of IL-17A inhibitor into the brain tissue, however, the expression of Beclin-1 increased significantly, along with the LC3II/LC3I ratio, while the expressions of P62, mTOR and PI3K-I protein decreased significantly. In terms of behavioral scores, the scores of optic neuritis and myelitis were more serious, onset occurred earlier and the progress was faster, after the administration of IL-17A. In the mechanism of NMO animal model, IL-17A may regulate autophagy and affect the disease process through the activation of the PI3K-mTOR signaling pathway.

Melatonin prevents sleep deprivation-associated anxiety-like behavior in rats: role of oxidative stress and balance between GABAergic and glutamatergic transmission.

Sleep deprivation (SD) has been shown to induce anxiety-like behavior. Melatonin, an endogenous potent antioxidant, protects neurons from oxidative stress in many disease models. Here we investigated the effect of melatonin against SD-induced anxiety-like behavior and attempted to define the possible mechanisms involved. SD was induced in rats using modified multiple platform model. Melatonin (15 mg/kg) was administered to the rats via intraperitoneal injection. The elevated plus maze test, open field test and light-dark exploration were used to evaluate anxiety-like behavior. Serum corticosterone was measured to determine stress level. Malondialdehyde (MDA) level and superoxide dismutase (SOD) enzyme activity of amygdala and serum were performed to determine the level of oxidative stress. Levels of protein were detected by means of Western blot. The results showed that SD induces anxiety-like behavior, while melatonin treatment prevented these changes. Serum corticosterone also increased with SD but its levels were normalized by melatonin. In addition, melatonin reversed SD-induced changes in MDA and SOD in both of amygdala and serum. The results of Western blot showed that melatonin attenuated the up-regulation of NR2B-containing N-methyl-D-aspartate receptors, GluR1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor as well as phosphorylation of GluR1 at Ser831, and Ca2+/calmodulin-dependent protein kinase II-alpha in SD rats. Meanwhile, melatonin blocked the down-regulation of γ-aminobutyric acid A-alpha-2 receptor. In conclusion, our results suggest that melatonin prevents anxiety-like behavior induced by SD. The possible mechanism may be attributed to its ability to reduce oxidative stress and maintain balance between GABAergic and glutamatergic transmission.

Characterization of a newly isolated strain Pseudomonas sp. C27 for sulfide oxidation: Reaction kinetics and stoichiometry.

Sulfide biooxidation by the novel sulfide-oxidizing bacteria Pseudomonas sp. C27, which could perform autotrophic and heterotrophic denitrification in mixotrophic medium, was studied in batch and continuous systems. Pseudomonas sp. C27 was able to oxidize sulfide at concentrations as high as 17.66 mM. Sulfide biooxidation occurred in two distinct stages, one resulting in the formation of sulfur with nitrate reduction to nitrite, followed by thiosulfate formation with nitrite reduction to N2. The composition of end-products was greatly impacted by the ratio of sulfide to nitrate initial concentrations. At a ratio of 0.23, thiosulfate represented 100% of the reaction products, while only 30% with a ratio of 1.17. In the continuous bioreactor, complete removal of sulfide was observed at sulfide concentration as high as 9.38 mM. Overall sulfide removal efficiency decreased continuously upon further increases in influent sulfide concentrations. Based on the experimental data kinetic parameter values were determined. The value of maximum specific growth rate, half saturation constant, decay coefficient, maintenance coefficient and yield were to be 0.11 h(-1), 0.68 mM sulfide, 0.11 h(-1), 0.21 mg sulfide/mg biomass h and 0.43 mg biomass/mg sulfide, respectively, which were close to or comparable with those reported in literature by other researches.

Protective Effects of Gastrodin Against Autophagy-Mediated Astrocyte Death.

Gastrodin is an active ingredient derived from the rhizome of Gastrodia elata. This compound is usually used to treat convulsive illness, dizziness, vertigo, and headache. This study aimed to investigate the effect of gastrodin on the autophagy of glial cells exposed to lipopolysaccharides (LPS, 1 µg/mL). Autophagy is a form of programmed cell death, although it also promotes cell survival. In cultured astrocytes, LPS exposure induced excessive autophagy and apoptosis, which were significantly prevented by the pretreatment cells with gastrodin (10 µM). The protective effects of gastrodin via autophagy inhibition were verified by the decreased levels of LC3-II, P62, and Beclin-1, which are classical markers for autophagy. Furthermore, gastrodin protected astrocytes from apoptosis through Bcl-2 and Bax signaling pathway. The treatment of astrocytes with rapamycin (500 nM), wortmannin (100 nM), and LY294002 (10 µM), which are inhibitors of mTOR and PI3K, respectively, eliminated the known effects of gastrodin on the inhibited Beclin-1 expression. Furthermore, gastrodin blocked the down-regulation of glutamine synthetase induced by LPS exposure in astrocytes. Our results suggest that gastrodin can be used as a preventive agent for the excessive autophagy induced by LPS.

Anxiolytic effects of sesamin in mice with chronic inflammatory pain.

OBJECTIVE: Sesamin is known for its role in antioxidant, antiproliferative, antihypertensive, and neuroprotective activities. However, little is known about the role of sesamin in the development of emotional disorders. Here we investigated persistent inflammatory pain hypersensitivity and anxiety-like behaviors in the mouse suffering chronic pain. METHODS: Chronic inflammatory pain was induced by hind paw injection of complete Freund's adjuvant (CFA). Levels of protein were detected by Western blot. RESULTS: Administration of sesamin could induce anxiolytic activities but had no effect on analgesia. In the basolateral amygdala, a structure involving the anxiety development, sesamin attenuated the up-regulation of NR2B-containing N-methyl-d-aspartate receptors, GluR1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor as well as phosphorylation of GluR1 at Ser831 (p-GluR1-Ser831), and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII-alpha) in the hind paw CFA-injected mice. In the same model, we found that the sesamin blocked the down-regulation of gamma-aminobutyric acid A (GABAA-alpha-2) receptors. CONCLUSION: Our findings show that sesamin reduces anxiety-like behaviors induced by chronic pain at least partially through regulating the GABAergic and glutamatergic transmission in the amygdala of mice.

Neuroprotective effects of a novel translocator protein (18 kDa) ligand, ZBD-2, against focal cerebral ischemia and NMDA-induced neurotoxicity.

Ligands of the translocator protein (18 kDa) (TSPO) have demonstrated rapid anxiolytic efficacy in stress responses and stress-related disorders. This protein is involved in the synthesis of endogenous neurosteroids including pregnenolone, dehydroepiandrosterone, and progesterone. These neurosteroids promote γ-aminobutyric acid-mediated neurotransmission in the central neural system (CNS). A TSPO ligand, N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2) was recently synthesized. The purpose of the present study was to investigate the neuroprotective effects of ZBD-2 and. In cultured cortical neurons, treatment with ZBD-2 attenuated excitotoxicity induced by N-methyl-d-aspartate (NMDA) exposure. It significantly decreased the number of apoptotic cells by downregulating GluN2B-containing NMDA receptors (NMDARs), the ratio of Bax/Bcl-2, and levels of pro-caspase-3. Systemic treatment of ZBD-2 provided significant neuroprotection in mice subjected to middle cerebral artery occlusion. These findings provide direct evidence that neuroprotection by ZBD-2 is partially mediated by inhibiting GluN2B-containing NMDA receptor-mediated excitotoxicity.

Anxiolytic-like effects of translocator protein (TSPO) ligand ZBD-2 in an animal model of chronic pain.

The activation of Translocator protein (18 kDa) (TSPO) has been demonstrated to mediate rapid anxiolytic efficacy in stress response and stress-related disorders. This protein is involved in the synthesis of endogenous neurosteroids that promote γ-aminobutyric acid (GABA)-mediated neurotransmission in the central neural system. However, little is known about the functions and the underlying mechanisms of TSPO in chronic pain-induced anxiety-like behaviors. The novel TSPO ligand N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2) was used in the present study. We found that ZBD-2 (0.15 or 1.5 mg/kg) significantly attenuated anxiety-like behaviors in mice with chronic inflammatory pain induced by hindpaw injection of complete Freund's adjuvant (CFA). However, the treatment did not alter the nociceptive threshold or inflammation in the hindpaw. Hindpaw injection of CFA induced the upregulation of TSPO, GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and NR2B-containing N-methyl-D-aspartate (NMDA) receptors in the basolateral amygdala (BLA). ZBD-2 administration reversed the alterations of the abovementioned proteins in the BLA of the CFA-injected mice. Electrophysiological recording revealed that ZBD-2 could prevent an imbalance between excitatory and inhibitory transmissions in the BLA synapses of CFA-injected mice. Therefore, as the novel ligand of TSPO, ZBD-2 induced anxiolytic effects, but did not affect the nociceptive threshold of mice under chronic pain. The anxiolytic effects of ZBD-2 were related to the regulation of the balance between excitatory and inhibitory transmissions in the BLA.

MicroRNA-494 sensitizes colon cancer cells to fluorouracil through regulation of DPYD.

Chemoresistance of colon cancer cells to the chemotherapeutics is still a main obstacle in treatment of this malignancy. The microRNA (miRNA) mediated chemosensitivity regulation in colon cancer cells is still largely unknown. Here we constructed a fluorouracil (5-Fu) resistant SW480 cell line (SW480/5-Fu) and discovered that miRNA miR-494 was down-regulated in the drug resistant cells compared with the parental cells. miR-494 level was found to be correlated with 5-Fu sensitivity in colon cancer cells, and artificial alteration of miR-494 affects the sensitivity of colon cancer cell lines to 5-Fu. miR-494 also promoted apoptosis of colon cancer cells at present of 5-Fu. Importantly, as a regulatory enzyme in the 5-Fu catabolic pathway, DPYD was confirmed to be a direct target of miR-494 through the interaction of miR-494 and its binding site within DPYD 3' untranslated region (3'UTR). miR-494 also negatively regulated endogenous DPYD expression in SW480 cells. Overexpression or knockdown of DPYD could attenuate miR-494 mediated 5-Fu sensitivity regulation, suggesting the dependence of DPYD regulation in miR-494 activity. miR-494 inhibited SW480/5-Fu derived xenograft tumors growth in vivo at present of 5-Fu. Thus, we concluded that in colon cancer cells, tumor suppressor miR-494 enhanced 5-Fu sensitivity via regulation of DPYD expression.

Bioreactor performance and functional gene analysis of microbial community in a limited-oxygen fed bioreactor for co-reduction of sulfate and nitrate with high organic input.

Limited-oxygen mediated synergistic relationships between sulfate-reducing bacteria (SRB), nitrate-reducing bacteria (NRB) and sulfide-oxidizing bacteria (SOB, including nitrate-reducing, sulfide-oxidizing bacteria NR-SOB) were predicted to simultaneously remove contaminants of nitrate, sulfate and high COD, and eliminate sulfide generation. A lab-scale experiment was conducted to examine the impact of limited oxygen on these oxy-anions degradation, sulfide oxidation and associated microbial functional responses. In all scenarios tested, the reduction of both nitrate and sulfate was almost complete. When limited-oxygen was fed into bioreactors, S(0) formation was significantly improved up to ∼ 70%. GeoChip 4.0, a functional gene microarray, was used to determine the microbial gene diversity and functional potential for nitrate and sulfate reduction, and sulfide oxidation. The diversity of the microbial community in bioreactors was increased with the feeding of limited oxygen. Whereas the intensities of the functional genes involved in sulfate reduction did not show a significant difference, the abundance of the detected denitrification genes decreased in limited oxygen samples. More importantly, sulfide-oxidizing bacteria may alter their populations/genes in response to limited oxygen potentially to function more effectively in sulfide oxidation, especially to elemental sulfur. The genes fccA/fccB from nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB), such as Paracoccus denitrificans, Thiobacillus denitrificans, Beggiatoa sp., Thiomicrospira sp., and Thioalkalivibrio sp., were more abundant under limited-oxygen condition.

Kinetics of nitrate and sulfate removal using a mixed microbial culture with or without limited-oxygen fed.

The biological degradation of nitrate and sulfate was investigated using a mixed microbial culture and lactate as the carbon source, with or without limited-oxygen fed. It was found that sulfate reduction was slightly inhibited by nitrate, since after nitrate depletion the sulfate reduction rate increased from 0.37 mg SO4 (2-)/mg VSS d to 0.71 mg SO4 (2-)/mg VSS d, and the maximum rate of sulfate reduction in the presence of nitrate corresponded to 56 % of the non-inhibited sulfate reduction rate determined after nitrate depleted. However, simultaneous but not sequential reduction of both oxy-anions was observed in this study, unlike some literature reports in which sulfate reduction starts only after depletion of nitrate, and this case might be due to the fact that lactate was always kept above the limiting conditions. At limited oxygen, the inhibited effect on sulfate reduction by nitrate was relieved, and the sulfate reduction rate seemed relatively higher than that obtained without limited-oxygen fed, whereas kept almost constant (0.86-0.89 mg SO4 (2-)/mg VSS d) cross the six ROS states. In contrast, nitrate reduction rates decreased substantially with the increase in the initial limited-oxygen fed, showing an inhibited effect on nitrate reduction by oxygen. Kinetic parameters determined for the mixed microbial culture showed that the maximum specific sulfate utilization rate obtained (0.098 ± 0.022 mg SO4 (2-)/(mg VSS h)) was similar to the reported typical value (0.1 mg SO4 (2-)/(mg VSS h)), also indicating a moderate inhibited effect by nitrate.

[Clinical observation of visual quality after aspherical intraocular lens implantation in traumatic cataract].

OBJECTIVE: To observe the visual quality of aspherical intraocular lens (IOL) implantation in traumatic cataract patients. METHODS: Prospective clinical study.96 traumatic cataract patients (96 eyes) suffered from penetrating corneal trauma chosen from the first affiliated hospital of Zhengzhou university during June 2009 to June 2012. They were divided into two groups based on the different type of intraocular lens. The experimental group (48 eyes) was implanted with aspherical IOL and the control group (48 eyes) was implanted with traditional sphere IOL.Uncorrected visual acuity (UCVA), best corrected visual acuity (BCVA), contrast sensitivity (CS) and stereoscopic vision were observed at 1 month, 3 months, and 6 months after surgery. At the same time, questionnaire survey about the satisfaction of patients was also performed. The t test was used to compare the preoperative general condition, postoperative visual acuity, contrast sensitivity and stereoscopic vision of the two groups, and the rank sum test was used to compare the astigmatism and the satisfaction of patients. RESULTS: There was no significant difference in UCVA (t = 1.37, 1.28,0.71, P > 0.05) between the experimental group (0.56 ± 0.22, 0.68 ± 0.13,0.84 ± 0.15) and the control group (0.51 ± 0.17, 0.61 ± 0.20,0.81 ± 0.17) at three time points. There was no significant difference in BCVA (t = 0.87, 1.38, 1.39, P > 0.05) between the experimental group (0.62 ± 0.13, 0.74 ± 0.21, 0.87 ± 0.10) and the control group (0.57 ± 0.25,0.69 ± 0.22,0.84 ± 0.15) . The same result happened in stereoscopic vision at 6 months after surgery (far stereopsis:123.5 ± 7.8 vs 126. 9 ± 5.9, t = 0.64, P > 0.05;near stereopsis:90.5 ± 7.8 vs 95.2 ± 3.5; t = 1.36, P > 0.05) between experimental group and control group. The contrast sensitivity of the experimental group in every stage (3 c/d:1.52 ± 0.18, 6 c/d:1.68 ± 0.19, 12 c/d:1.29 ± 0.14, 18 c/d:1.04 ± 0.20) was superior to the control group (3 c/d:1.49 ± 0.27, 6 c/d:1.57 ± 0.21, 12 c/d:1.14 ± 0.20, 18 c/d:0.85 ± 0.14) , especially on the glare sensitivity (the experimental group:3 c/d:1.40 ± 0.15, 6 c/d:1.52 ± 0.22, 12 c/d:1.21 ± 0.18, 18 c/d:0.91 ± 0.14, the control group:3 c/d:1.13 ± 0.13, 6 c/d:1.13 ± 0.28, 12 c/d:0.92 ± 0.13, 18 c/d:0.54 ± 0.16) Compared two groups of difference have statistical significance (free from glare:3 c/d:t = 2.829, 6 c/d:t = 4.092, 12 c/d:t = 3.055, 18 c/d:t = 2.093;glare:3 c/d:t = 2.650, 6 c/d:t = 3.105, 12 c/d:t = 3.395, 18 c/d:t = 2.215;P < 0.05) .Questionnaire survey showed the experimental group (72.9%) was statistically significantly higher (t = 3.016, P < 0.05) than that in the control group (54.1%) on the satisfaction of patients. CONCLUSIONS: The visual quality with implantation of aspherical IOL in traumatic cataract patients is superior to traditional sphere IOL. Aspherical IOL is more appropriate for the patients with small and peripheral corneal scar.It can reduce the visual function damage to minimum caused by trauma.

[Quantitative observation on changes of anterior segment by ultrasound biomicroscopy after posterior chamber phakic intraocular lens implantation].

OBJECTIVE: The objective is to study the safety and effectiveness of implantation of posterior chamber phakic intraocular contact lens (ICL) by observing the changes in anterior segment using ultrasound biomicroscopy (UBM). METHODS: It was a perspective study. The study sampled 30 high myopia patients (30 eyes) who were treated with posterior chamber phakic ICL implant. Central anterior chamber depth (ACD), trabecular-iris angle (TIA), angle opening distance (AOD500), trabecular-ciliary processes distance (TCPD) and iris-ciliary processes distance (ICPD) were measured using UBM preoperatively, 3 months and 1 year postoperatively. The distance from ICL to the central surface of lens and peripheral lens and intra-ocular pressure were measured postoperatively and examined using slit-lamp biomicroscope. One-way ANOVA was used to analyze the distance between peripheral surface of ICL and the lens. One-way repeated measures ANOVA and Bonferroni were conducted. RESULTS: Preoperatively, 3 months and 1 year postoperatively, ACD were (3.16±0.08) mm, (2.76±0.13) mm, (2.74±0.14) mm; AOD500 were (0.45±0.04) mm, (0.41±0.04) mm, (0.41±0.03) mm; TIA were (35.00±3.24)°, (32.47±3.56)°, (32.40±3.23)°, respectively. There were significant difference in TIA, ACD and AOD (P<0.05) between preoperative and postoperative data. There was no significant difference between the two postoperative periods tested. TCPD and ICPD showed no significant difference between various time points (F=0.49, F=0.57; P>0.05). CONCLUSIONS: The decrease in ACD depth and correction in TIA and AOD were the noticeable changes observed in morphological structure of the ocular anterior segment after the ICL treatment. The incidence of complication did not increase as the result of the minor changes in morph structure during the course of the study. However, the long-term effects would require further long-term observation.

[Manganese superoxide dismutase gene transfection of mouse small intestinal epithelial cells protects them from radiation injury].

OBJECTIVE: To investigate the protective effect of manganese superoxide dismutase (MnSOD) gene transfer to small intestinal epithelial cells from radiation injury. METHODS: Herpes simplex virus (HSV) vector containing both the human MnSOD and GFP genes was introduced into mouse small intestine. Expression of MnSOD by the intestinal villi was confirmed by nested RT-PCR, immunofluorescence and enzyme activity assay. Mice were then given various doses of irradiation over the abdomen. The height of intestinal villi was measured on histopathology sections by SZ-PT optical system before irradiation, 24 h and 72 h post-irradiation. All comparisons were performed by one-way analysis of variance using the SPSS statistical software to analyze the significance between groups. RESULTS: Nested RT-PCR, immunofluorescence and enzyme activity assay of MnSOD demonstrated overexpression and increased activity of MnSOD in the inoculated intestine of mice. Control (sham inoculated) irradiated mice showed decreased villi height by 40.1%-59.3% on day 1 and 44.2%-65.1% on day 3 (7.5-15 Gy). Treatment of mice with HSV-MnSOD prior to radiation led to statistically significant radioprotection of the small bowel with mean villi height decreased by only 3.1%-12.4% on day 1 and 6.3%-29.1% on day 3. CONCLUSION: The results demonstrate that overexpression of human MnSOD via a replication defective herpes simplex viral vector is an effective method to protect the small intestine from damage caused by ionizing radiation.

Gene transfer of human manganese superoxide dismutase protects small intestinal villi from radiation injury.

Small bowel toxicity represents a major dose-limiting side effect of radiation treatment for many malignancies. We examined the effects of overexpressing human manganese superoxide dismutase (MnSOD) in the small intestine in mice to prevent radiation enteritis. Mice were treated with the human MnSOD gene delivered enterally using a nontoxic, replication-defective herpes simplex virus (HSV)-1-based vector. HSV vectors containing the human MnSOD transgene and green fluorescent protein (GFP) transgene, or GFP transgene alone, were constructed and injected intraluminally into a 2cm length of small intestine of C3H/HeNsd mice. Total body irradiation of 15 Gy was delivered to mice inoculated 24 hours earlier with either HSV-MnSOD (10(3) to 10(8) plaque-forming units), control HSV-GFP, or no vector. At 24 or 72 hours after irradiation, mice were killed and villi areas were measured from appropriate segments of the small intestine. Control irradiated mice showed a decreased villi area of 82% by day 3 after irradiation, whereas treatment of mice with HSV-MnSOD 10(8) plaque-forming units led to only a 16% decrease in villi area (P < 0.001) before radiation. Similar findings were seen on day 3 and were associated with a significant (P < 0.001) preservation of enteric protein content in HSV-MnSOD-treated mice. A dose-dependent effect of MnSOD in preventing radiation-induced small bowel injury was evident. These data demonstrate that overexpression of human MnSOD via a replication-defective herpes viral vector is an efficacious method of protecting the small intestine from ionizing radiation damage.