Efficient production of R-2-(4-hydroxyphenoxy) propionic acid by Beauveria bassiana using biofilm-based two-stage fermentation.

In this work, a novel biofilm-based fermentation of Beauveria bassiana was employed to convert R-2- phenoxypropionic acid (R-PPA) to R-2-(4-hydroxyphenoxy) propionic acid (R-HPPA). The biofilm culture model of Beauveria bassiana produced a significantly higher R-HPPA titer than the traditional submerged fermentation method. Mannitol dosage, tryptone dosage, and initial pH were the factors that played a significant role in biofilm formation and R-HPPA synthesis. Under the optimal conditions, the maximum R-HPPA titer and productivity approached 22.2 g/L and 3.2 g/(L·d), respectively. A two-stage bioreactor combining agitation and static incubation was developed to further increase R-HPPA production. The process was optimized to achieve 100 % conversion of R-PPA, with a maximum R-HPPA titer of 50 g/L and productivity of 3.8 g/(L·d). This newly developed biofilm-based two-stage fermentation process provides a promising strategy for the industrial production of R-HPPA and related hydroxylated aromatic compounds.

The AKT inhibitor, MK-2206, attenuates ABCG2-mediated drug resistance in lung and colon cancer cells.

Introduction: The overexpression of ATP-binding cassette (ABC) transporters, ABCB1 and ABCG2, are two of the major mediators of multidrug resistance (MDR) in cancers. Although multiple ABCB1 and ABCG2 inhibitors have been developed and some have undergone evaluation in clinical trials, none have been clinically approved. The compound, MK-2206, an inhibitor of the protein kinases AKT1/2/3, is undergoing evaluation in multiple clinical trials for the treatment of certain types of cancers, including those resistant to erlotinib. In this in vitro study, we conducted in vitro experiments to determine if MK-2206 attenuates multidrug resistance in cancer cells overexpressing the ABCB1 or ABCG2 transporter. Methodology: The efficacy of MK-2206 (0.03-1 µM), in combination with the ABCB1 transporter sub-strates doxorubicin and paclitaxel, and ABCG2 transporter substrates mitoxantrone, SN-38 and topotecan, were determined in the cancer cell lines, KB-C2 and SW620/Ad300, which overexpress the ABCB1 transporter or H460/MX20 and S1-M1-80, which overexpress the ABCG2 transporter, respectively. The expression level and the localization of ABCG2 transporter on the cancer cells membranes were determined using western blot and immunofluorescence assays, respectively, following the incubation of cells with MK-2206. Finally, the interaction between MK-2206 and human ABCG2 transporter was predicted using computer-aided molecular modeling. Results: MK-2206 significantly increased the efficacy of anticancer compounds that were substrates for the ABCG2 but not the ABCB1 transporter. MK-2206 alone (0.03-1 µM) did not significantly alter the viability of H460/MX20 and S1-M1-80 cancer cells, which overexpress the ABCG2 transporter, compared to cells incubated with vehicle. However, MK-2206 (0.3 and 1 µM) significantly increased the anticancer efficacy of mitoxantrone, SN-38 and topotecan, in H460/MX20 and S1-M1-80 cancer cells, as indicated by a significant decrease in their IC50 values, compared to cells incubated with vehicle. MK-2206 significantly increased the basal activity of the ABCG2 ATPase (EC50 = 0.46 µM) but did not significantly alter its expression level and sub-localization in the membrane. The molecular modeling results suggested that MK-2206 binds to the active pocket of the ABCG2 transporter, by a hydrogen bond, hydrophobic interactions and π-π stacking. Conclusion: These in vitro data indicated that MK-2206 surmounts resistance to mitoxantrone, SN-38 and topotecan in cancer cells overexpressing the ABCG2 transporter. If these results can be translated to humans, it is possible that MK-2206 could be used to surmount MDR in cancer cells overexpressing the ABCG2 transporter.

Effect of high-intensity ultrasound soymilk pretreatment on the physicochemical properties of microbial transglutaminase-catalyzed tofu gel.

Tofu prepared by conventional methods often has a bitter taste and poor water-holding capacity (WHC). To improve the quality of the product, alternative processes must be developed. Herein, the effect of ultrasound pretreatment on the properties of soymilk and tofu gel derived thereof were investigated. Treatment of soymilk with ultrasound gave rise to a reduction in the particle size and an enhancement in the surface hydrophobicity, whereby optimum values were obtained after 15 min treatment. Subsequently, microbial transglutaminase (MTG) was added to ultrasound-treated soymilk to promote the soy protein crosslinking. The gel strength, WHC, and nonfreezable water content of MTG-catalyzed tofu gel obtained from treated soymilk increased with the extension of the ultrasound pretreatment time, whereas the free sulfhydryl content decreased because of the formation of disulfide bonds. Fourier transform infrared spectroscopy demonstrated variations in the secondary structure of MTG-catalyzed tofu gel. Furthermore, soymilk's exposure to high-intensity ultrasound pretreatment led to a tofu gel with a dense, homogenous, and stable network structure, as evidenced by scanning electron microscopy. Therefore, this study answers for the theoretical support of the industrial production of MTG-catalyzed tofu gel from ultrasound-treated soymilk. PRACTICAL APPLICATION: High-intensity ultrasound pretreatment improved the texture properties of MTG-catalyzed tofu gel. The resulting MTG-catalyzed tofu gel has potential application in industrial production.

Sapitinib Reverses Anticancer Drug Resistance in Colon Cancer Cells Overexpressing the ABCB1 Transporter.

The efficacy of anti-cancer drugs in patients can be attenuated by the development of multi-drug resistance (MDR) due to ATP-binding cassette (ABC) transporters overexpression. In this in vitro study, we determined the reversal efficacy of the epidermal growth factor receptor (EFGR) inhibitor, saptinib, in SW620 and SW720/Ad300 colon cancer cells and HEK293/ABCB1 cells which overexpress the ABCB1 transporter. Sapitinib significantly increased the efficacy of paclitaxel and doxorubicin in ABCB1 overexpressing cells without altering the expression or the subcellular location of the ABCB1 transporter. Sapitinib significantly increased the accumulation of [3H]-paclitaxel in SW620/AD300 cells probably by stimulating ATPase activity which could competitively inhibit the uptake of [3H]-paclitaxel. Furthermore, sapitinib inhibited the growth of resistant multicellular tumor spheroids (MCTS). The docking study indicated that sapitinib interacted with the efflux site of ABCB1 transporter by π-π interaction and two hydrogen bonds. In conclusion, our study suggests that sapitinib surmounts MDR mediated by ABCB1 transporter in cancer cells.

In Vitro and In Vivo Evaluation of Olmesartan Medoxomil Microcrysta ls and Nanocrystals: Preparation, Characterization, and Pharmacokinet ic Comparison in Beagle Dogs.

BACKGROUND: Olmesartan medoxomil (OLM) is a promising prodrug hydrolyzed to olmesartan (OL) during absorption from the gastrointestinal tract. OL is a selective angiotensin II receptor antagonist, with high drug resistance and low drug interaction. However, OLM has low solubility and low bioavailability. Therefore, it is extremely urgent to reduce the drug particle size to improve its biological bioavailability. OBJECTIVE: The aim of the study was to improve the oral bioavailability of poorly water-soluble olmesartan medoxomil (OLM) by using different particle size-reduction strategies. METHOD: Raw drug material was micronized or nanosized by either jet or wet milling processes, respectively. The particle sizes of the prepared nanocrystals (100-300 nm) and microcrystals (0.5-16 µm) were characterized by DLS, SEM, and TEM techniques. Solid state characterization by XPRD and DSC was used to confirm the crystalline state of OLM after the milling processes. RESULTS: We demonstrated that OLM nanocrystals enhanced solubility and dissolution in the non-sink condition in which high sensitivity was found in purified water. After 1 h, 65.4% of OLM was dissolved from nanocrystals, while microcrystals and OLMETEC® only showed 37.8% and 31.9% of drug dissolution, respectively. In the pharmacokinetic study using Beagle dogs, an increase in Cmax (～2 fold) and AUC (～1.6 fold) was observed after oral administration of OLM nanocrystals when compared to microcrystals and reference tablets, OLMETEC®. In contrast, OLM microcrystals failed to improve the oral bioavailability of the drugs. CONCLUSION: Particles size reduction to nano-scale by means of nanocrystals technology significantly increased in vitro dissolution rate and in vivo oral bioavailability of OLM.

Glesatinib, a c-MET/SMO Dual Inhibitor, Antagonizes P-glycoprotein Mediated Multidrug Resistance in Cancer Cells.

Multidrug resistance (MDR) is one of the leading causes of treatment failure in cancer chemotherapy. One major mechanism of MDR is the overexpressing of ABC transporters, whose inhibitors hold promising potential in antagonizing MDR. Glesatinib is a dual inhibitor of c-Met and SMO that is under phase II clinical trial for non-small cell lung cancer. In this work, we report the reversal effects of glesatinib to P-glycoprotein (P-gp) mediated MDR. Glesatinib can sensitize paclitaxel, doxorubicin, colchicine resistance to P-gp overexpressing KB-C2, SW620/Ad300, and P-gp transfected Hek293/ABCB1 cells, while has no effect to their corresponding parental cells and negative control drug cisplatin. Glesatinib suppressed the efflux function of P-gp to [3H]-paclitaxel and it didn't impact both the expression and cellular localization of P-gp based on Western blot and immunofluorescent analysis. Furthermore, glesatinib can stimulate ATPase in a dose-dependent manner. The docking study indicated that glesatinib interacted with human P-gp through several hydrogen bonds. Taken together, c-Met/SMO inhibitor glesatinib can antagonize P-gp mediated MDR by inhibiting its cell membrane transporting functions, suggesting new application in clinical trials.

Ciprofloxacin Enhances the Chemosensitivity of Cancer Cells to ABCB1 Substrates.

ABCB1 is one of the major drug efflux transporters that is known to cause multidrug resistance (MDR) in cancer patients receiving chemotherapy for the treatment of solid tumors and hematological malignancies. Inhibition of ABCB1 efflux function is important for maintaining the intracellular concentration of chemotherapeutic drugs. Here, we evaluated ciprofloxacin for its ability to reverse MDR caused by the overexpression of ABCB1. Cytotoxicity of ciprofloxacin was determined by the MTT assay. The chemosensitizing effects of ciprofloxacin were determined in combination with ABCB1 substrates. The intracellular accumulation and efflux of ABCB1 substrates was measured by a scintillation counter, and protein expression was determined by the Western blotting. Vanadate-sensitive ATPase assay was performed to determine the effect of ciprofloxacin on the ATPase activity of ABCB1, and docking analysis was done to determine the interaction of ciprofloxacin with ABCB1. Ciprofloxacin significantly potentiated the cytotoxic effects of ABCB1 substrates in ABCB1-overexpressing cells. Furthermore, ciprofloxacin increased the intracellular accumulation and decreased the efflux of [³H]-paclitaxel without altering the expression of ABCB1. Ciprofloxacin stimulated the ATPase activity of ABCB1 in a concentration-dependent manner. Our findings showed that ciprofloxacin potently inhibits the ABCB1 efflux function and it has potential to be developed as a combination anticancer therapy.

Effect of raw material variability of glipizide on the in vitro dissolution rate and in vivo bioavailability performance: The importance of particle size.

The objective of this study was to understand the impact of active pharmaceutical ingredients (API) particle size on a re-developed generic product of glipizide and to improve its formulation so that it exhibits bioequivalent to that of the reference listed drug (RLD). Two commercial batches of APIs (API-1 and API-2) with the same polymorphism and one batch of home-made APIs (API-3) with super-small particle size were used in the present study. The in vitro dissolution profiles of the tested formulations were compared with the RLD in a series of dissolution media. Then, the impact of particle size on in vivo absorption was evaluated in Beagle dogs. Compared with the RLD, formulation A with larger API size showed slower dissolution in pH 6.0 and 7.4 medium, resulting bioinequivalent with the RLD. Conversely, formulation B with smaller API size demonstrated similar in vitro dissolution profiles with the RLD and thus exhibited bioequivalent in the present study. Furthermore, formulation C with super small particle size still exhibited identical oral absorption although rapid dissolution was observed in the tested condition. Herein, it indicated that 2-5 µm might be defined as the "inert size range" of glipizide for ensuring the bioequivalence with the RLD. The results in the present study might help to obtain a better understanding of the variability in raw materials for oral absorption, develop a bioequivalent product and thus post-market quality control.

Novel SS-31 modified liposomes for improved protective efficacy of minocycline against drug-induced hearing loss.

Hearing loss, which is regarded as a worldwide public health concern, lacks approved therapeutic strategies. Current drug candidates used to treat hearing loss commonly have low efficacy. To achieve the optimum drug efficacy, we designed a liposome system to preload a clinically approved, water-soluble drug, minocycline. Inspired by our previous research, we used a mitochondria-targeting tetrapeptide, SS-31, to modify the surface of liposomes. The results revealed that SS-31 modified, minocycline-loaded liposomes significantly increased hair cell survival against chronic exposure to gentamicin in a zebrafish model. The designed formulation maintained the activity of mechanotransduction channels in the hair cells, and thus did not result in any alteration in gentamicin uptake. This suggested that the protective efficacy of the liposomes was induced by modulating targets associated with cell death. Further studies are required to clarify the exact intracellular mechanism of the designed formulation and to determine its clinical benefits in patients with hearing dysfunction.

Optimization and evaluation of lipid emulsions for intravenous co-delivery of artemether and lumefantrine in severe malaria treatment.

Parenteral therapy for severe and complicated malaria is necessary, but currently available parenteral antimalarials have their own drawbacks. As for recommended artemisinin-based combination therapy, antimalarial artemether and lumefantrine are limited in parenteral delivery due to their poor water solubility. Herein, the aim of this study was to develop the lipid-based emulsions for intravenous co-delivery of artemether and lumefantrine. The lipid emulsion was prepared by high-speed shear and high-pressure homogenization, and the formulations were optimized mainly by monitoring particle size distribution under autoclaved conditions. The final optimal formulation was with uniform particle size distribution (~ 220 nm), high encapsulation efficiency (~ 99%), good physiochemical stability, and acceptable hemolysis potential. The pharmacokinetic study in rats showed that Cmax of artemether and lumefantrine for the optimized lipid emulsions were significantly increased than the injectable solution, which was critical for rapid antimalarial activity. Furthermore, the AUC0-t of artemether and lumefantrine in the lipid emulsion group were 5.01- and 1.39-fold of those from the solution, respectively, suggesting enhanced bioavailability. With these findings, the developed lipid emulsion is a promising alternative parenteral therapy for the malaria treatment, especially for severe or complicated malaria.

A sensitive, high-throughput, and ecofriendly method for the determination of lumefantrine, artemether, and its active metabolite dihydroartemisinin by supercritical fluid chromatography and tandem mass spectrometry.

A quick and sensitive supercritical fluid chromatography with tandem mass spectrometry method for the simultaneous determination of lumefantrine, artemether, and its active metabolite dihydroartemisinin in rat plasma was developed and validated. The chromatographic separation was performed on an ACQUITY UPC2 ™ BEH 2-EP column within 2.5 min by gradient elution using compressed CO2 and methanol containing 2 mM ammonium acetate as the mobile phases. Detection was achieved by multiple reaction monitoring using electrospray ionization in the positive ionization mode. For sample preparation, 50 µL of the sample was processed by modified high-throughput, one-step protein precipitation using hydrogen peroxide as a stabilizer to protect the endoperoxide-containing artemisinin derivatives from degradation. The calibration curves were linear over the concentration range of 2.0-1000 ng/mL for both artemether and dihydroartemisinin, and 1.0-5000 ng/mL for lumefantrine. The values of selectivity, lower limit of quantification, linearity, accuracy, precision, matrix effects, stability, and recovery met the acceptable range according to the Food and Drug Administration guidelines. The developed method enables high resolution and speed as well as low cost, low solvent consumption, and short time and was successfully applied to pharmacokinetic studies through the intravenous administration of an artemether-lumefantrine lipid emulsion in rats.

Screening the expression characteristics of several miRNAs in G93A-SOD1 transgenic mouse: altered expression of miRNA-124 is associated with astrocyte differentiation by targeting Sox2 and Sox9.

MicroRNAs (miRNAs) are suspected to be a contributing factor in amyotrophic lateral sclerosis (ALS). Here, we assess the altered expression of miRNAs and the effects of miR-124 in astrocytic differentiation in neural stem cells of ALS transgenic mice. Differentially expressed miRNA-positive cells (including miR-124, miR-181a, miR-22, miR-26b, miR-34a, miR-146a, miR-219, miR-21, miR-200a, and miR-320) were detected by in situ hybridization and qRT-PCR in the spinal cord and the brainstem. Our results demonstrated that miR-124 was down-regulated in the spinal cord and brainstem. In vitro, miR-124 was down-regulated in neural stem cells and up-regulated in differentiated neural stem cells in G93A-superoxide dismutase 1 (SOD1) mice compared with WT mice by qRT-PCR. Meanwhile, Sox2 and Sox9 protein levels showed converse change with miR-124 in vivo and vitro. After over-expression or knockdown of miR-124 in motor neuron-like hybrid (NSC34) cells of mouse, Sox2 and Sox9 proteins were noticeably down-regulated or up-regulated, whereas Sox2 and Sox9 mRNAs remained virtually unchanged. Moreover, immunofluorescence results indicated that the number of double-positive cells of Sox2/glial fibrillary acidic protein (GFAP) and Sox9/glial fibrillary acidic protein (GFAP) was higher in G93A-SOD1 mice compared with WT mice. We also found that many Sox2- and Sox9-positive cells were nestin positive in G93A-SOD1 mice, but not in WT mice. Furthermore, differentiated neural stem cells from G93A-SOD1 mice generated a greater proportion of astrocytes and lower proportion of neurons than those from WT mice. MiR-124 may play an important role in astrocytic differentiation by targeting Sox2 and Sox9 in ALS transgenic mice. Cover Image for this issue: doi: 10.1111/jnc.14171.

[The influence of joining central venous catheter and pressure transducer with T-junctions on central venous pressure].

OBJECTIVE: To investigate the influence of the number of T-junctions between central venous catheter and pressure transducer on measurement of central venous pressure ( CVP ) in patients. METHODS: A prospective controlled study was conducted. The patients with CVP monitoring in Department of Critical Care Medicine of the Fifth Center Hospital in Tianjin from February to October in 2014 were enrolled. The patients were divided into three groups according to the number of T-junction between central venous catheter and pressure transducer: without T-junction control group and 1, 2, 3 T-junctions groups. In each patient, corresponding CVP values with different number of T-junctions placed between the central venous catheter and pressure sensors were determined within a certain period, and a square-wave graphic was obtained and preserved on the monitor. The own frequency ( fn ) and the attenuation coefficient ( D ) of the system of pressure measurement were calculated after measurement of the shock wave following a square-wave to obtain the distance between two vibrations and the amplitude of the shock wave. The difference in CVP, fn and D were compared among the groups. RESULTS: A total of 20 cases were enrolled, and 150 groups of data were collected. (1) With the increase in the number of T-junction, CVP showed a tendency of gradual reduction. The CVP of the groups of control and 1, 2, 3 T-junctions was ( 7.00±1.60 ), ( 7.00±3.00 ), ( 5.00±2.00 ), and ( 4.00±1.00 ) mmHg ( 1 mmHg = 0.133 kPa ), respectively. The CVP of 3 T-junctions group was significantly lower than that of the control group ( F = 9.333, P = 0.015 ). (2) With an increase in the number of T-junction, fn showed a tendency of gradual increase. The fn of groups control and 1, 2, 3 T-junctions was ( 12.30±0.79 ), ( 16.00±0.91 ), ( 18.10±1.75 ), ( 20.90±2.69 ) Hz, respectively. The fn of 1, 2, 3 T-junctions group was significantly higher than that of the control group ( F1 = 45.962, F2 = 45.414, F3 = 46.830, all P = 0.000 ); the fn of groups 2 and 3 T-junctions was significantly higher than that of 1 T-junction group ( F1= 5.827, P1= 0.042; F2 = 15.038, P2 = 0.004 ), but there was no significant difference between the groups of 2 T-junctions and 3 T-junctions ( F = 3.800, P = 0.087 ). (3) With an increase of the number of T-junction, D also showed a tendency of gradual increase. The D of 1, 2, 3 T-junction group was 1.62±0.27, 1.60±0.22, 1.82±0.25, and 2.15±0.58, respectively. There were no differences among four groups. CONCLUSIONS: After the application of T-junctions between central venous catheter and pressure transducer, CVP values will be underestimated, the reason of which is considered to be the increase in length and thinner lumen of the T-junctions.

A chimeric vacuolar Na(+)/H(+) antiporter gene evolved by DNA family shuffling confers increased salt tolerance in yeast.

The vacuolar Na(+)/H(+) antiporter plays an important role in maintaining ionic homeostasis and the osmotic balance of the cell with the environment by sequestering excessive cytoplasmic Na(+) into the vacuole. However, the relatively low Na(+)/H(+) exchange efficiency of the identified Na(+)/H(+) antiporter could limit its application in the molecular breeding of salt tolerant crops. In this study, DNA family shuffling was used to create chimeric Na(+)/H(+) antiporters with improved transport activity. Two homologous Na(+)/H(+) antiporters from halophytes Salicornia europaea (SeNHX1) and Suaeda salsa (SsNHX1) were shuffled to generate a diverse gene library. Using a high-throughput screening system of yeast complementation, a novel chimeric protein SseNHX1 carrying 12 crossover positions and 2 point mutations at amino acid level was selected. Expression of SseNHX1 in yeast mutant exhibited approximately 46% and 22% higher salt tolerance ability in yeast growth test than that of SsNHX1and SeNHX1, respectively. Measurements of the ion contents demonstrated that SseNHX1 protein in yeast cells accumulated more Na(+) and slightly more K(+) than the parental proteins did. Furthermore, this chimera also conferred increased tolerance to LiCl and a similar tolerance to hygromycin B compared with the parental proteins in yeast.

Hydrophilic C terminus of Salicornia europaea vacuolar Na(+)/H(+) antiporter is necessary for its function.

Plant vacuolar Na(+)/H(+) antiporters play important roles in cellular ion homeostasis,vacuolar pH regulation and sequestration of Na(+) ions into the vacuole. Previous research showed that hydrophilic C-terminal region of Arabidopsis AtNHX1 negatively regulates the Na(+)/H(+) transporting activity. In this study, we truncated the hydrophilic C terminus of a vacuolar Na(+) /H(+) antiporter gene from Salicornia europaea (SeNHX1) to generate its derivative, SeNHX1-ΔC. Expression of SeNHX1 and SeNHX1-ΔC in yeast mutant showed that SeNHX1 significantly improved the tolerance to NaCl; however, the expression of SeNHX1-ΔC enormously decreased the tolerance to NaCl. Overall, these results suggest that the hydrophilic C-terminal region of SeNHX1 is required for Na(+)/H(+) exchanging activity of SeNHX1.

Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobacco.

To evaluate the physiological importance of cytosolic ascorbate peroxidase (APX) in the reactive oxygen species (ROS)-scavenging system, a full-length cDNA clone, named LmAPX, encoding a cytosolic ascorbate peroxidase was isolated from Lycium chinense Mill. using homologous cloning, then the expression of LmAPX under salt stress was investigated. After sequencing and related analysis, the LmAPX cDNA sequence was 965 bp in length and had an open reading frame (ORF) of 750 bp coding for 250 amino acids. Furthermore, the LmAPX sequence was sub-cloned into prokaryotic expression vector pET28a and the recombinant proteins had a high expression level in Escherichia coli. Results from a southern blot analysis indicated that three inserts of this gene existed in the tobacco genome encoding LmAPX. Compared with the control plants (wild-type and empty vector control), the transgenic plants expressing the LmAPX gene exhibited lower amount of hydrogen peroxide (H2O2) and relatively higher values of ascorbate peroxidase activity, proline content, and net photosynthetic rate (Pn) under the same salt stress. These results suggested that overexpression of the LmAPX gene could decrease ROS production caused by salt stress and protect plants from oxidative stress.

miRNA-9 expression is upregulated in the spinal cord of G93A-SOD1 transgenic mice.

The pathogenesis of amyotrophic lateral sclerosis (ALS) remains unclear. Accumulating evidence indicates that various miRNAs expressed in a spatially and temporally controlled manner in the nervous system have an important function in the development of neurodegenerative diseases. The present study aimed to determine the expression and cellular distribution of miRNA-9 in the spinal cord of G93A-SOD1 mutant mice at different time points (post-natal 95, 108 and 122 d). miRNA expression was evaluated by microarray analysis; differentially expressed miRNAs were validated by RT-qPCR. The cellular distribution of miRNA-9 was analyzed by in-situ hybridization. Microarray results indicated for the first time that various miRNAs were differentially expressed between the G93A-SOD1 mutant mice and the littermate control mice. miRNA-9 expression was upregulated at 95, 108, and 122 d as validated by microarray analysis, RT-qPCR, and ISH. ISH results also showed that the miRNA-9-positive cells mainly expressed in the cytoplasm were located in the dorsal horn and the ventral horn of the spinal cord. The majority of miRNA-9-positive cells were located in the ventral horn of the gray matter, the locus of neurodegeneration. These results indicated that the differential expression of miRNA-9 may have an important function in the pathogenesis of G93A-SOD1 transgenic mice.

[Correlation of D-amino acid-oxidase gene polymorphism to schizophrenia].

OBJECTIVE: To investigate the genetic association between schizophrenia and polymorphism of D-amino acid-oxidase (DAAO) gene. METHODS: A total of 112 parent/offspring trios in which the proband met the Amerecan Classification and Diagnostic Criteria for Mental Disorders (Fourth Revised Edition) were included in this study. Correlation analysis between schizophrenia and DAAO gene polymorphism and haplotype relative risk analysis were conducetd by using PCR and SNP typing in all the nuclear families. RESULTS: The rs3918347 allele was correlated to schizophrenia (P = 0.014). Allele A was a protective factor (Z = -2.37) and allele G the hazard factor (Z = 2.37). The frequency of rs3918347 allele A was 0.41 and that of the allele G was 0.59. The rs3741775, rs3825251 and rs4964770 alleles were not associated with schizophrenia. Three haplotypes of C/G in the rs3825251-rs3918347, G/T in the rs3918347-rs4964770, C/G/T in the rs3825251-rs3918347-rs4964770 were associated with schizophrenia (P = 0.021, 0.036, and 0.028, with genotype frequencies of 0.33, 0.28, and 0.15, respectively). CONCLUSION: The nucleotide polymorphism of DAAO gene is associated with schizophrenia in Chinese population.