MicroRNA miR-18a-3p promotes osteoporosis and possibly contributes to spinal fracture by inhibiting the glutamate AMPA receptor subunit 1 gene (GRIA1).

The promoting role that miR-18a-3p plays in osteoporosis (OP) has been previously described. However, the detailed mechanisms remain unclear. Bone tissues were collected from healthy patients, OP patients, and patients with osteoporotic spinal fractures. An osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) was constructed to detect the expression of miR-18a-3p and glutamate AMPA receptor subunit 1 (GRIA1). Alkaline phosphatase (ALP) activity and a qRT-PCR analysis were used to detect ALP content, alizarin red S staining was used to detect calcium deposition, and qRT-PCR was used to evaluate runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) expression levels. A dual-luciferase reporter and RNA pull-down assay was used to verify the targeted correlation between miR-18a-3p and GRIA1. We observed an increase in miR-18a-3p expression and a decrease in GRIA1 expression in OP and osteoporotic vertebral fracture patients. Upregulation of miR-18a-3p restrained the activity and expression of ALP in hBMSCs, inhibited the expression of RUNX2, OCN, and OPN, and inhibited calcium deposition. Knockdown of miR-18a-3p or upregulation of GRIA1 promoted osteogenic differentiation. Our findings indicate that miR-18a-3p promotes OP progression by regulating GRIA1 expression, suggesting that targeting miR-18a-3p/GRIA1 may be a therapeutic strategy for OP.

Knockdown of H19 Attenuates Ox-LDL-induced Vascular Smooth Muscle Cell Proliferation, Migration, and Invasion by Regulating miR-599/PAPPA Axis.

ABSTRACT: Long noncoding RNAs could participate in the development of atherosclerosis (AS). However, the underlying mechanism by which long noncoding RNA H19 is implicated in AS remains largely unknown. In this study, we investigated the function of H19 on cell proliferation, migration, and invasion in oxidized low-density lipoprotein (ox-LDL)-treated human aortic vascular smooth muscle cells (HA-VSMCs), and on hyperlipidemia response in high-fat diet (HFD)-treated ApoE-/- mice. Moreover, we explored the target interaction among H19, microRNA (miR)-599, and pappalysin 1 (PAPPA). Our results showed that H19 expression was elevated in serum samples of patients with AS and ox-LDL-treated HA-VSMC. H19 silence mitigated ox-LDL-induced proliferation, migration, and invasion of HA-VSMCs. H19 acted as a sponge for miR-599, and miR-599 knockdown reversed the suppressive effect of H19 silence on proliferation, migration, and invasion of HA-VSMCs. PAPPA was a target of miR-599 and attenuated the inhibitive role of miR-599 in HA-VSMC processes. H19 knockdown repressed PAPPA expression by increasing miR-599. Moreover, H19 interference alleviated hyperlipidemia response in HFD-treated ApoE-/- mice. Collectively, knockdown of H19 inhibited proliferation, migration, and invasion of ox-LDL-treated HA-VSMCs and hyperlipidemia response in HFD-treated ApoE-/- mice by regulating miR-599/PAPPA axis, indicating H19 might act as a potential target for the treatment of AS.

LncRNA XIST knockdown ameliorates oxidative low-density lipoprotein-induced endothelial cells injury by targeting miR-204-5p/TLR4.

Oxidative low-density lipoprotein (ox-LDL)-induced endothelial cell injury is a key contributor to atherosclerosis development. However, the role and mechanism of long noncoding RNA X-inactive specific transcript (XIST) in atherosclerosis remain largely unknown. The ox-LDL-induced human umbilical vein endothelial cells (HUVECs) injury was analyzed by cell viability, apoptosis, inflammatory cytokines secretion and oxidative stress. The expression levels of XIST, microRNA-204-5p (miR-204-5p) and toll-like receptor 4 (TLR4) were detected by quantitative real-time polymerase chain reaction and western blot, respectively. The target interaction between miR-204-5p and XIST or TLR4 was explored by bioinformatics analysis, luciferase assay and RNA immunoprecipitation. The expression of XIST was enhanced in ox-LDL-treated HUVECs. Knockdown of XIST attenuated ox-LDL-induced viability inhibition, apoptosis production, inflammatory response and oxidative stress in HUVECs. XIST was validated as a sponge of miR-204-5p and TLR4 acted as a target of miR-204-5p. Knockdown of miR-204-5p reversed silence of XISTmediated suppressive role in ox-LDL-induced injury. TLR4 alleviated miR-204-5p-mediated inhibitive effect on ox-LDL-induced injury. Moreover, XIST could regulate TLR4 expression by sponging miR-204-5p. In conclusion, silence of XIST displayed a protective role in ox-LDL-induced injury in HUVECs by regulating miR-204-5p/TLR4 axis, providing a novel mechanism for understanding the pathogenesis of atherosclerosis.

An agarase of glycoside hydrolase family 16 from marine bacterium Aquimarina agarilytica ZC1.

A novel ß-agarase gene aga672 was cloned from strain ZC1, the typical strain of agar-degrading Aquimarina agarilytica. Gene aga672 is composed of 2130 bp, and the encoded protein Aga672 showed an amino acid sequence identity of only 42% with reported agarases. Aga672 should belong to glycoside hydrolase family 16 according to the protein sequence similarity. The molecular mass of the recombinant Aga672 was estimated to be 98 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Aga672 decomposed agarose to produce neoagarotetraose, neoagarohexaose and neoagarooctaose as the main products. That is the main difference between Aga672 and other reported agarases of family GH16. The Km and Vmax for agarose degradation were 59.8 mg mL-1 and 154.3 U mg-1, respectively. The activity of Aga672 was stable at temperatures below 40°C and at pH 7.0-11.0 with the maximal agarase activity at 25°C and pH 7.0. The results showed that agarase Aga672 could be suitable to hydrolyze the gelated agarose. Thus, it has potential applications in the production of neoagarooligosaccharides directly from red alga.

[HPLC combined with PCA technology for analysis of five gingerol compounds in different processing degrees of ginger charcoal].

To establish a new method for simultaneously determining the content of five gingerol compounds in different processing degrees of ginger charcoal and PCA principal component analysis was conducted for analysis. Samples were analyzed on Ultimate TM XB-C18 column (4.6 mm x 250 mm, 5 µm) , with acetonitrile (A) -0.1% phosphoric acid solution (B) as mobile phase for gradient elution. Detection wavelength was set at 280 nm. The flow rate was 0.6 mL x min(-1) and the column temperature was 30 degrees C. The five compounds were separated well and showed good linearity (r ≥ 0.999 7) within the concentration ranges tested. The average value for recoveries was between 98.86% - 101.5% (RSD 1.4% - 2.9%). The contents of five compounds showed difference among different processing degrees of ginger charcoal. Zingiberone had the highest content in the standard carbon, and the content of gingerol was decreased as the deepening of processing degree. Different processing degrees of ginger charcoal were classified into three groups with PCA, and provided scientific basis for establishing the quality standards of ginger charcoal.

Characterization of a novel ß-agarase from an agar-degrading bacterium Catenovulum sp. X3.

An agar-degrading bacterium, Catenovulum sp. X3, was isolated from the seawater of Shantou, China. A novel ß-agarase gene agaXa was cloned from the strain Catenovulum sp. X3. The gene agaXa consists of 1,590 bp and encodes a protein of 529 amino acids, with only 40 % amino acid sequence identity with known agarases. AgaXa should belong to the glycoside hydrolase family GH118 based on the amino acid sequence similarity. The molecular mass of the recombinant AgaXa (rAgaXa) was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had a maximal agarase activity at 52 °C and pH 7.4 and was stable over pH 5.0 ~ 9.0 and at temperatures below 42 °C. The K m and V max for agarose were 10.5 mg/ml and 588.2 U/mg, respectively. The purified rAgaXa showed endolytic activity on agarose degradation, yielding neoagarohexaose, neoagarooctaose, neoagarodecaose, and neoagarododecaose as the end products. The results showed that AgaXa has potential applications in agar degradation for the production of oligosaccharides with various bioactivities.

Gene cloning, expression and characterization of a neoagarotetraose-producing ß-agarase from the marine bacterium Agarivorans sp. HZ105.

A ß-agarase gene hz2 with 2,868 bp was cloned from the marine agarolytic bacterium Agarivorans sp. HZ105. It encoded a mature agarase HZ2 of 102,393 Da (920 amino acids). Based on the amino acid sequence similarity, agarase HZ2 was assigned to the glycoside hydrolase family 50. The ß-agarase shared a gene sequence identity of 98.6% with the reported but much less characterized ß-agarase agaB from Vibrio sp. JT0107. Its recombinant agarase rHZ2 was produced in E. coli cells and purified to homogeneity. The agarase rHZ2 degraded agarose and neoagarooligosaccharides with degrees of polymerization above four, to yield neoagarotetraose as the dominant product, which was different from ß-agarase agaB of Vibrio sp. JT0107. The agarose hydrolysis pattern suggested that rHZ2 was an endo-type ß-agarase. Beta-mercaptoethanol (90 mM) and dithiothreitol (9 mM) increased the agarase activity of rHZ2 by 72.9% and 17.3% respectively, while SDS (9 mM) inhibited the activity completely. The agarase activity was independent of Na(+), K(+), Mg(2+) and Ca(2+). The maximal enzyme activity was observed at 40°C and pH 7. The kinetic parameters K (m), V (max), K (cat), and K (cat)/K (m) values toward agarose of agarase rHZ2 were 5.9 mg ml(-1), 235 U mg(-1), 401 s(-1) and 6.8 × 10(5) M(-1) s(-1), respectively. Agarase rHZ2 could have a potential application in the production of bioactive neoagarotetraose.

[Primary study on quality standard of carbonizing drug characteristic of ginger carbon].

OBJECTIVE: To establish the quality standard for carbonizing drug characteristic of ginger carbon. METHOD: Gingers and different carbonized gingers were compared by the absorption of pigment, tannin content, pH, mouth's coagulation time and bleeding time. RESULT: The study resulted in the recommended carbonizing standard that the absorption capacity shall not be less than 7.50 mg x g(-1) for methylene blue, the tannin content shall not be less than 2.103 mg x g(-1), the pH shall be (5.50 +/- 0.10), and coagulation time and bleeding time shall be the shorter the better. CONCLUSION: The established assessment standard for carbonizing drug characteristic of ginger carbon is reasonable, easily operated and feasible.

Draft genome sequence of the novel agarolytic bacterium Aquimarina agarilytica ZC1.

The marine bacterium ZC1 is the type strain of the recently identified novel species Aquimarina agarilytica. It can produce multiple agarases. Here we report the draft genome sequence of strain ZC1 (4,253,672 bp, with a GC content of 32.8%) and major findings from its annotation. It is the first reported genome in the genus Aquimarina.

Aquimarina agarilytica sp. nov., an agarolytic species isolated from a red alga.

A novel yellow-pigmented, agarolytic bacterial strain, designated ZC1T, was isolated from the surface of the marine red alga Porphyra haitanensis collected near Nan Ao Island, Guangdong province, China. The isolate was Gram-stain-negative, strictly aerobic and rod-shaped and displayed ß-galactosidase, alkaline phosphatase, catalase and oxidase activities. The predominant cellular fatty acids were iso-C15:0, summed feature 3 (comprising C16:1ω7c and/or iso-C15:0 2-OH) and iso-C17:0 3-OH. The major menaquinone was menaquinone 6 (MK-6). The DNA G+C content was 32.8 mol%. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain ZC1T was closely related to members of the genus Aquimarina in the family Flavobacteriaceae, phylum Bacteroidetes. Based on phylogenetic and phenotypic evidence, strain ZC1T (=CCTCC AB 2010229T=NBRC 107695T) represents the type strain of a novel species in the genus Aquimarina, for which the name Aquimarina agarilytica sp. nov. is proposed.

[Survey on human rabies cases and its viral molecular biological features in Baoshan city, Yunnan province].

OBJECTIVE: To understand the epidemiological features of two rabies cases in Baoshan city year 2006 and 2007 and to analyze its source of infection. METHODS: Questionnaires were used to do the epidemiological survey on each of the rabies cases. Brain tissue samples of rabies patients were collect to detect the rabies virus by direct immunofluorescence assay (DFA) and RT-PCR assay. Homology and phylogenetic tree were analyzed, based on the whole nucleotide and deduced amino acid sequence of P, M and N gene of rabies virus followed by molecular epidemiological analysis. RESULTS: In July 2006, one human rabies case was identified in Longyang district, and another one in Tengchong county in Baoshan city in 2007. The degrees of exposure of these two patients was all at degree III. Two brain tissue samples among the dead patients (No. CYN0601H and CYN0701H) were confirmed positive by both DFA and RT-PCR assay. The homology analysis of P, M and N gene sequences among CYN0601H, CYN0701H and other rabies strains isolated from other provinces and other counties, showed that the samples in Baoshan city shared the highest homology with the strains in Thailand. Phylogenetic analysis indicated that the two samples were very close and all belonged to genetype 1 Lyssavirus, with the closest relationship between samples in Baoshan city and strains in Thailand. CONCLUSION: It was confirmed on the virus molecular level that the two patients in Baoshan city were both suffered from rabies. The prevalent strains in Baoshan city was probably imported from foreign country, suggesting that prevention and control measures on rabies virus in the boarder areas of Yunnan should be strengthened.