Objective: To study the effects of downregulation of osteopontin (OPN) on biological behaviors and matrix metalloproteinase-2 (MMP-2) expression of breast cancer cells MDA-MB-231. Methods: siRNA expression vectors were designed to target OPN and named as pSilence-OPN S 1 and pSilence-OPN S 2. The two plasmids were transfected into MDA-MB-231 cells. The mRNA and protein expression of OPN and MMP-2 were measured by RT-PCR and Western blotting, respectively. The effect of downregulation of OPN on cell proliferation, cell cycle distribution, and malignant phenotype of MDA-MB-231 cells were determined by MIT assay, flow cytometry, and soft agar assay, respectively. The level of MMP-2 protein in the supernatant of cultured cells was measured with ELISA. Results: The expression of OPN in the transfected cells (M-OPN S 1 and M-OPN S 2) was significantly downregulated at both mRNA and protein levels compared with MDA-MB-231 cells. The proliferation of transfected cells was inhibited and the cells were arrested at S phase. Downregulation of OPN caused a significant reduction in colony formation rate and MMP-2 expression. Conclusions: The stable silencing of OPN gene expression with siRNA inhibited the proliferation of MDA-MB-231 cells, downregulats malignant degree and MMP-2 expression. It may provide a novel therapeutic approach for breast cancer therapy.

AIM:To observe the structural basis of ocular motility abnormalities in patients with congenital fibrosis of the extraocular muscles type Ⅰ ( CFEOM Ⅰ) due to missense mutations in the developmental kinesin KIF21A using high - resolution magnetic resonance imaging ( MRI) .
METHODS: Totally 11 affected individuals reported KIF21A mutations were correlated with MRI studies demonstrating extraocular muscles ( EOMs ) size, location, contractility, and innervation. EOMs and the motor nerve in the orbits were imaged with T1 weighted in a triplanar scan using a dual-phased coils with 2. 0mm thick. Motor nerves were imaged at the brainstem using head coils and 3D-FIESTA with 0. 6-mm thick.
RESULTS: Patients with CFEOM Ⅰ exhibited different degrees of hypoplasia of oculomotor nerve, the abducens nerve and the trochlear nerve were also affected, of which 8 cases of orbital section could see the signal of abnormal nerve dominated by oculomotor nerve to lateral rectus. The both sides of six EOMS in all patients exhibited variable atrophy and abnormal bright internal signal on T1 imaging, particularly severe for the superior rectus and levator muscles.
CONCLUSION: High - resolution MRI can directly demonstrate pathology of motor nerves,affected EOMs, and ‘Pulley' hypoplasia caused by CFEOM Ⅰ due to mutations in KIF21A,and these findings suggest that the neuronal hypoplasia is the etiological factor of CFEOM.

Spermatogenesis originates from spermatogonial stem cells (SSCs). SSCs can continually renew and eventually differentiate into spermatozoa under control of various growth factors, microenvironments and self-signaling. The molecules involved in SSCs self-renewal include glial cell-derived nerve growth factor, fibroblast growth factor and downstream signaling pathways, as well as the transcription factors and the epigenetic regulators. Molecules related to SSCs differentiation include retinoic acid, a variety of transcription factors and the eptigenetic regulatory factors. The research on the mechanism of SSCs self-renewal and differentiation is of great significance for the understanding of spermatogenesis and the diagnosis and treatment of male infertility. This review summarized the exogenous factors, transcription factors, and epigenetic regulators that are involved in the regulation of SSCs self-renewal and differentiation in recent years.

BACKGROUNDReactive oxygen species (ROS) may play both physiological and pathophysiological roles. Transcription factor NF-E2-related factor 2 (Nrf2) regulates antioxidant response element (ARE)-mediated genes expression and coordinates induction of chemoprotective proteins in response to physical and chemical stresses. The exact role of Nrf2 in cellular responses to different levels of oxidative stresses remains unknown.

METHODSRat pulmonary microvascular endothelial cells were cultured and treated with 0 mmol/L, 0.125 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 1.0 mmol/L and 2.0 mmol/L hydrogen peroxide solution for 2 hours. Nrf2 gene expression was assayed by reverse transcription-PCR, Nrf2-ARE binding activity was assayed with electrophoretic mobility shift assay (EMSA), and localization of Nrf2 was detected with immunohistochemistry.

RESULTSLow and moderate (0.125 mmol/L, 0.25 mmol/L and 0.5 mmol/L) doses hydrogen peroxide exposure of rat pulmonary microvascular endothelial cells led to the nuclear accumulation of Nrf2, increased activity of transcription regulation and up-regulation of ARE-medicated gene expression. In contrast, high doses of hydrogen peroxide (1 mmol/L, 2 mmol/L) exposure of the cells led to the nuclear exclusion of Nrf2, decreased activity transcription regulation and down-regulation of ARE-mediated gene expression.

CONCLUSIONLow and moderate doses of hydrogen peroxide play protective roles by increasing transcription activity of Nrf2, whereas high- dose hydrogen peroxide plays a deleterious role by decreasing transcription activity of Nrf2.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Cells, Cultured ; Electrophoretic Mobility Shift Assay ; Gene Expression ; drug effects ; Hydrogen Peroxide ; pharmacology ; Immunohistochemistry ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction

Summer school is an important reform of the teaching organization mode home and abroad. The purpose is to make the best use of the limited teaching resources to meet the needs of the students. In recent years, a lot of universities has accumulated abundant experiences to set up summer school in China. This article focuses on the practice of summer school in Beijing University of Chinese Medicine, in order to provide some promoting strategies for summer school in domestic universities of Traditional Chinese Medicine in future.

Objective To explore the inhibitory effect of matrix metalloproteinase-2(MMP-2) gene silencing in vitro and in vivo on the invasion and growth of laryngeal cancer cells. Methods siRNA recombinant lentivirus targeting MMP-2 gene was transfected into Hep-2 cells, and MMP-2 protein expression was analyzed consequently by using western-blot. Invasive properties of transfectants were evaluated by Boyden assay. In addition, the lentivirus was intratumorally injected in a model of the grafted nude mouse and the morphological changes of transfectants were examined by transmission electron microscope. Finally, cell proliferation in xenografts was measured by immunolabeling of proliferating cell nuclear antigen (PCNA). Results Over 90% of target cancer cells were found to be transfected by MMP-2-RNAi-Lentivirus. Western-blot analysis revealed that none of transfectants expressed MMP-2 protein whereas most untreated cancer cells exhibited positive protein expression. Significant differences were found between the treated and untreated groups regarding the number of trans fectants penetrating through an artificial basement in a Boyden chamber(12±4 vs 35±6, x±s, t = 14. 492, P < 0. 01), and the average value of weight [(1.186±0.225)g vs [(2.127±0.344)g] and volume [(0.974±0.216) cm<'3> vs (1.618±0.272) cm3] of the grafted tumors (t was 7.094 and 5.684,P <0.01). The overall tumor inhibitive rate was about 44. 2%. Transmission electron microscope showed an obviously decreased invasive feature of transfectants. Finally, the percentages of transfectants inununolabeled for PCNA were significantly lower in the treated group (49. 588±6. 995) than those (71. 434±7. 043) in control one (t = 9. 573, P < 0. 01). Condusions The invasion, growth and proliferation of laryngeal cancer can be inhibited by siRNA mediated MMP-2 gene silencing. These data strongly suggest that MMP-2 gene silencing by siRNA technology could be a promising approach to cancer therapy.

We sought to identify common key regulators and build a gene-metabolite network in different nonalcoholic fatty liver disease (NAFLD) phenotypes. We used a high-fat diet (HFD), a methionine-choline-deficient diet (MCDD) and streptozocin (STZ) to establish nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH) and NAFL+type 2 diabetes mellitus (T2DM) in rat models, respectively. Transcriptomics and metabolomics analyses were performed in rat livers and serum. A functional network-based regulation model was constructed using Cytoscape with information derived from transcriptomics and metabolomics. The results revealed that 96 genes, 17 liver metabolites and 4 serum metabolites consistently changed in different NAFLD phenotypes (>2-fold, P<0.05). Gene-metabolite network analysis identified ccl2 and jun as hubs with the largest connections to other genes, which were mainly involved in tumor necrosis factor, P53, nuclear factor-kappa B, chemokine, peroxisome proliferator activated receptor and Toll-like receptor signaling pathways. The specifically regulated genes and metabolites in different NAFLD phenotypes constructed their own networks, which were mainly involved in the lipid and fatty acid metabolism in HFD models, the inflammatory and immune response in MCDD models, and the AMPK signaling pathway and response to insulin in HFD+STZ models. Our study identified networks showing the general and specific characteristics in different NAFLD phenotypes, complementing the genetic and metabolic features in NAFLD with hepatic and extra-hepatic manifestations.
AMP-Activated Protein Kinases ; Animals ; Complement System Proteins ; Diabetes Mellitus ; Diet ; Diet, High-Fat ; Insulin ; Liver ; Metabolism ; Metabolomics ; Models, Animal ; Non-alcoholic Fatty Liver Disease* ; Peroxisomes ; Phenotype ; Rats ; Streptozocin ; Toll-Like Receptors ; Tumor Necrosis Factor-alpha

BACKGROUNDRecently, due to the rapid development of proteomic techniques, great advance has been made in many scientific fields. We aimed to use magnetic beads (liquid chip) based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technology to screen distinctive biomarkers for lung adenocarcinoma (adCA), and to establish the diagnostic protein profiles.

METHODSUsing weak cation exchange magnetic beads (MB-WCX) to isolate and purify low molecular weight proteins from sera of 35 lung adCA, 46 benign lung diseases (BLDs) and 44 healthy individuals. The resulting spectra gained by anchor chip-MALDI-TOF-MS were analyzed by ClinProTools and a pattern recognition genetic algorithm (GA).

RESULTSIn the working mass range of 800 - 10 000 Da, 99 distinctive peaks were resolved in lung adCA versus BLDs, while 101 peaks were resolved in lung adCA versus healthy persons. The profile gained by GA that could distinguish adCA from BLDs was comprised of 4053.88, 4209.57 and 3883.33 Da with sensitivity of 80%, specificity of 93%, while that could separate adCA from healthy control was comprised of 2951.83 Da and 4209.73 Da with sensitivity of 94%, specificity of 95%. The sensitivity provided by carcinoembryonic antigen (CEA) in this experiment was significantly lower than our discriminatory profiles (P < 0.005). We further identified a eukaryotic peptide chain release factor GTP-binding subunit (eRF3b) (4209 Da) and a complement C3f (1865 Da) that may serve as candidate biomarkers for lung adCA.

CONCLUSIONMagnetic beads based MALDI-TOF-MS technology can rapidly and effectively screen distinctive proteins/polypeptides from sera of lung adCA patients and controls, which has potential value for establishing a new diagnostic method for lung adCA.

Adenocarcinoma ; blood ; diagnosis ; Adult ; Aged ; Female ; Humans ; Lung Neoplasms ; blood ; diagnosis ; Magnetics ; Male ; Microspheres ; Middle Aged ; Sensitivity and Specificity ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods

OBJECTIVETo investigate the effect of iron on the growth, physiology and photosynthesis of cyanobacteria.

METHODSA gradient of iron concentrations was employed to investigate the growth, photo-pigments (chlorophyll A and phycocyanin), and cell chemical contents (C, N, P) of Microcystis aeruginosa in response to different iron additions.

RESULTSThe specific growth rate during the exponential growth phase, as well as the cell chlorophyll A and the phycocyanin content, was limited by iron below 12.3 tmol Fe x L(-1). The growth was inhibited when the iron concentration was at 24.6 micromol Fe x L(-1). The cell chlorophyll A and the phycocyanin content were saturated when the iron concentration was above 12.3 micromol Fe x L(-1) and declined slightly at 24.6 micromol Fe x L(-1). At a low iron concentration (about 6.15 micromol Fe x L(-1) and less), the cell nitrogen and carbohydrate content were iron limited, and the variation of the cell phosphorus content was similar to that of the nitrogen and carbohydrate, with a transition point of 12.3 micromol Fe x L(-1).

CONCLUSIONThe variation of cynobacteria growth is synchronous with that of the photo-pigments or the cell chemical content, and there exist relationships among photosynthesis, growth and internal chemical content, which could be useful for the growth estimation from the cell characteristics.

Carbohydrates ; analysis ; Culture Media ; chemistry ; Dose-Response Relationship, Drug ; Iron ; pharmacology ; Microcystis ; chemistry ; cytology ; drug effects ; physiology ; Nitrogen ; analysis ; Phosphorus ; analysis

Epididymitis is a commonly diagnosed disease associated with male infertility. However, little is known about the molecules that are involved in its development. This study was to identify critical genes associated with lipopolysaccharide-induced epididymitis and analyze the molecular mechanism of epididymitis through RNA sequencing. Experimental epididymitis models were generated by administering male Sprague-Dawley rats' lipopolysaccharide. A total of 1378 differentially expressed genes, including 531 upregulated and 847 downregulated genes, were identified in the epididymitis model rats compared with those in sham-operated rats by RNA sequencing. Functional enrichment analyses suggested that the upregulated genes were markedly enriched in inflammation-related biological processes, as well as in the tumor necrosis factor (TNF) signaling pathway, cytokine-cytokine receptor interactions, complement and coagulation cascades, and in the chemokine signaling pathway. Four downregulated genes (collagen type XXVIII alpha 1 chain [Col28α1], cyclin-dependent kinase-like 1 [Cdkl1], phosphoserine phosphatase [Psph], and fatty acid desaturase 2 [Fads2]) and ten upregulated genes (CCAAT/enhancer-binding protein beta [Cebpβ], C-X-C motif chemokine receptor 2 [Cxcr2], interleukin 11 [Il11], C-C motif chemokine ligand 20 [Ccl20], nuclear factor-kappa-B inhibitor alpha [Nfkbiα], claudin 4 [Cldn4], matrix metallopeptidase 9 [Mmp9], heat shock 70 kDa protein 8 [Hspa8], intercellular cell adhesion molecule-1 [Icam1], and Jun) were successfully confirmed by real-time polymerase chain reaction. Western blot demonstrated that CDKL1 was decreased, while MMP9 and NFKBIA were increased in the experimental model group compared with those in the sham-operated group. Our study sheds new light on the understanding of the early response of the epididymis during bacterial epididymitis.
Animals ; Base Sequence/genetics* ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Epididymis/metabolism* ; Epididymitis/metabolism* ; Gene Expression Profiling ; Genes/genetics* ; Lipopolysaccharides/pharmacology* ; Male ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, RNA