Airong Qian

Function of HAb18G/CD147 in Invasion of Host Cells by Severe Acute Respiratory Syndrome Coronavirus

Abstract To identify the function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome (SARS) coronavirus (CoV), we analyzed the protein-protein interaction among HAb18G/CD147, cyclophilin A (CyPA), and SARS-CoV structural proteins by coimmunoprecipitation and surface plasmon resonance analysis. Although none of the SARS-CoV proteins was found to be directly bound to HAb18G/CD147, the nucleocapsid (N) protein of SARS-CoV was bound to CyPA, which interacted with HAb18G/CD147. Further research showed that HAb18G/CD147, a transmembrane molecule, was highly expressed on 293 cells and that CyPA was integrated with SARS-CoV. HAb18G/CD147–antagonistic peptide (AP)–9, an AP of HAb18G/CD147, had a high rate of binding to 293 cells and an inhibitory effect on SARS-CoV. These results show that HAb18G/CD147, mediated by CyPA bound to SARS-CoV N protein, plays a functional role in facilitating invasion of host cells by SARS-CoV. Our findings provide some evidence for the cytologic mechanism of invasion by SARS-CoV and provide a molecular basis for screening anti-SARS drugs

Development of a Ground-Based Simulated Experimental Platform for Gravitational Biology

The purpose of this study is to develop a ground-based experimental platform which can provide a long-term, persistent, and stable weightless environment to provide technical support for the fundamental study of space life sciences. The platform included a superconducting magnet which can generate a large gradient high magnetic field, temperature control system, object stage, observing system, and gas control system. The platform can accurately measure and control comprehensive parameters, such as temperature, humidity, sample location, gravity level, and magnetic induction intensity. The biological experiment by means of the platform indicated that diamagnetic levitation affected cell morphology but had nonlethal effects on cell growth, which is consistent with the results of spaceflight. It suggests that the platform can meet the need of the simulated experimental conditions for gravitational biology research from molecule, cell, tissue, and organ to model animal.

INHIBITORY EFFECTS OF A GRADIENT STATIC MAGNETIC FIELD ON NORMAL ANGIOGENESIS

Angiogenesis, the formation of new blood vessels, is critical in many normal and pathological processes such as development, reproduction, tumor growth, and metastasis. Recently, exposure to moderate-intensity static magnetic fields (1 mT to 1 T) has attracted much attention for its potential therapeutic value as a noninvasive intervening method. Nevertheless, the effects of moderate-intensity and spatial gradient static magnetic fields (GSMF) on angiogenesis have not received enough attention. In this study, the effects of GSMF (0.2-0.4 T, 2.09 T/m, 1-11 days) on angiogenesis were investigated both in vitro and in vivo. An MTT assay was used as an in vitro method to detect the proliferation ability of human umbilical veins endothelial cells (HUVECs). Two kinds of in vivo models, a chick chorioallantoic membrane (CAM) and a matrigel plug, were used to detect the effects of GSMF on angiogenesis. The results showed that the proliferation ability of HUVECs was significantly inhibited 24 h after the onset of exposure. With regard to the CAM model, vascular numbers in the CAM that was continuously exposed to the GSMF were all less than those in normal condition. In accordance with the gross appearance, the contents of hemoglobin in the models exposed to GSMF for 7-9 days were also less. In addition, similar to the CAM model, the results of vascular density and hemoglobin contents in the matrigel plug also demonstrated that the GSMF exposure for 7 or 11 days inhibited vascularization. These findings indicate that GSMF might inhibit or prevent new blood vessels formation and could be helpful for the treatment of some diseases relevant to pathological angiogenesis.

Fractal Dimension as a Measure of Altered Actin Cytoskeleton in MC3T3-E1 Cells Under Simulated Microgravity Using 3-D/2-D Clinostats

Osteoblasts, the bone-forming cells, respond to various mechanical forces, such as stretch and fluid shear force in essentially similar ways. The cytoskeleton, as the load-bearing architecture of the cell, is sensitive to altered inertial forces. Disruption of the cytoskeleton will result in alteration of cellular structure and function. However, it is difficult to quantitatively illustrate cytoskeletal rearrangement because of the complexity of cytoskeletal structure. Usually, the morphological changes in actin organization caused by external stimulus are basically descriptive. In this study, fractal dimensions (D) analysis was used to quantify the morphological changes in the actin cytoskeleton of osteoblast-like cells (MC3T3-E1) under simulated microgravity using 3-D/2-D clinostats. The ImageJ software was used to count the fractal dimension of actin cytoskeleton by box-counting methods. Real-time PCR and immunofluroscent assays were used to further confirm the results obtained by fractal dimension analysis. The results showed significant decreases in D value of actin cytoskeleton, β-actin mRNA expression, and the mean fluorescence intensity of F-actin in osteoblast-like cells after 24 or 48 h of incubation under 3-D/2-D clinorotation condition compared with control. The findings indicate that 3-D/2-D clinorotation affects both actin cytoskeleton architecture and mRNA expression, and fractal may be a promising approach for quantitative analysis of the changes in cytoskeleton in different environments.

Whole-Body Vibration Effects on Bone Before and After Hind-Limb Unloading in Rats

INTRODUCTION Skeletal unloading during spaceflight leads to bone loss. Following long-duration flight, such loss may be very slow or impossible to fully recover. Therefore, it is important to seek countermeasures to prevent the loss. METHODS We studied the effects on bone of whole-body vibration (WBV) with variable parameters (10-60 Hz and 0.1-1 g) using hind-limb unloading (HLU) in the rat as a model for microgravity. The bone mineral densities (BMD) of the femur, tibia, and lumbar spines were measured and the mechanical properties of the femur were determined by mechanical testing. Serum alkaline phosphatase (ALP) and pyridinoline (PYD) levels were analyzed. RESULTS After 28 d of HLU, WBV reduced the losses of BMD in the femur and tibia from 18.8 to 10.1% and from 16.7 to 7.1%, respectively, and prevented the loss in stiffness of the femur after HLU. However, WBV had no effect on the lumbar spine. ALP levels were suppressed by HLU and maintained by WBV (28.3% increase). There was no significant difference in PYD among groups. In the recovery period following HLU, there were no significant effects of WBV on BMD, and the elastic modulus of the femur returned to normal. DISCUSSION AND CONCLUSIONS Mechanical stimulation in the form of WBV limited reduction of bone density when it was applied during the unloading, but not afterward. It is of interest that the mechanical properties of the femur were influenced by WBV in the control femurs as well as in the unloaded bones of this rat model.

Structure Prediction: New Insights into Decrypting Long Noncoding RNAs

Long noncoding RNAs (lncRNAs), which form a diverse class of RNAs, remain the least understood type of noncoding RNAs in terms of their nature and identification. Emerging evidence has revealed that a small number of newly discovered lncRNAs perform important and complex biological functions such as dosage compensation, chromatin regulation, genomic imprinting, and nuclear organization. However, understanding the wide range of functions of lncRNAs related to various processes of cellular networks remains a great experimental challenge. Structural versatility is critical for RNAs to perform various functions and provides new insights into probing the functions of lncRNAs. In recent years, the computational method of RNA structure prediction has been developed to analyze the structure of lncRNAs. This novel methodology has provided basic but indispensable information for the rapid, large-scale and in-depth research of lncRNAs. This review focuses on mainstream RNA structure prediction methods at the secondary and tertiary levels to offer an additional approach to investigating the functions of lncRNAs.

Large Gradient High Magnetic Fields Affect Osteoblast Ultrastructure and Function by Disrupting Collagen I or Fibronectin/αβ1 Integrin

The superconducting magnet generates a field and field gradient product that can levitate diamagnetic materials. In this study a specially designed superconducting magnet with a large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels (μ-g, 1-g, and 2-g), was used to simulate a space-like gravity environment. The effects of LG-HMF on the ultrastructure and function of osteoblast-like cells (MG-63 and MC3T3-E1) and the underlying mechanism were investigated by transmission electromicroscopy (TEM), MTT, and cell western (ICW) assays. Under LG-HMF significant morphologic changes in osteoblast-like cells occurred, including expansion of endoplasmic reticulum and mitochondria, an increased number of lysosomes, distorted microvilli, and aggregates of actin filaments. Compared to controls, cell viability and alkaline phosphatase (ALP) secretion were significantly increased, and collagen I (col I), fibronectin (FN), vinculin, integrin α3, αv, and β1 expression were changed under LG-HMF conditions. In conclusion, LG-HMF affects osteoblast ultrastructure, cell viability, and ALP secretion, and the changes caused by LG-HMF may be related to disrupting col I or FN/αβ1 integrin.

Diamagnetic Levitation Causes Changes in the Morphology, Cytoskeleton, and Focal Adhesion Proteins Expression in Osteocytes

Diamagnetic levitation technology is a novel simulated weightless technique and has recently been applied in life-science research. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels, namely, μg (diamagnetic levitation), 1g, and 2g for diamagnetic materials. In this study, the effects of LG-HMF on the activity, morphology, and cytoskeleton (actin filament, microtubules, and vimentin intermediate filaments) in osteocyte - like cell line MLO-Y4 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods, hematoxylin-eosin (HE) staining, and laser scanning confocal microscopy (LSCM), respectively. The changes induced by LG-HMF in distribution and expression of focal adhesion (FA) proteins, including vinculin, paxillin, and talin in MLO-Y4 were determined by LSCM and Western blotting. The results showed that LG-HMF produced by superconducting magnet had no lethal effects on MLO-Y4. Compared to control, diamagnetic levitation (μg) affected MLO-Y4 morphology, nucleus size, cytoskeleton architecture, and FA proteins distribution and expression. The study indicates that osteocytes are sensitive to altered gravity and FA proteins (vinculin, paxillin, and talin) may be involved in osteocyte mechanosensation. The diamagnetic levitation may be a novel ground-based space-gravity simulator and can be used for biological experiment at cellular level.

Large gradient high magnetic field affects FLG29.1 cells differentiation to form osteoclast-like cells.

Abstract Purpose: We aimed to investigate the effects of different apparent gravities (μ g, 1 g and 2 g) produced by large gradient high magnetic field (LGHMF) on human preosteoclast FLG29.1 cells. Materials and methods: FLG29.1 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. Cells were exposed to LGHMF for 72 h. On culture day 1, 2, 3, cell proliferation was detected by 3-(4,5)-dimethylthiahi-azo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method. On day 3, cell apoptosis and necrosis were assayed by Hoechst and propidium iodide (PI) staining. After cells were exposed to LGHMF for 72 h with the induction of 12-o-tetradecanoylphorbol 13-acetate (TPA), Tartrate-Resistant Acid Phosphatase (TRAP) positive cells and nitric oxide (NO) release were detected by TRAP staining and Griess method, respectively. Intracellular TRAP activity was measured using nitrophenylphosphate (pNPP) as the substrate. Results: MTT detection revealed that compared to control, FLG 29.1 cell proliferation in the μ g and 2 g groups were promoted. However, there is no obvious difference between the 1 g and control groups. Hoechst-PI staining showed that LGHMF promoted cell apoptosis and necrosis, especially in the 2 g group. Exposure to LGHMF inhibited the NO concentration of supernatant. Both the TRAP activity and the number of TRAP positive cells were higher in cells of μ g group than those in 2 g group. In the 1 g group, they were decreased significantly compared to control. Conclusions: These findings indicate that LGHMF could directly affect human preosteoclast FLG29.1 cells survival and differentiation. High magnetic flux inhibited osteoclasts formation and differentiation while reduced apparent gravity enhanced osteoclastogenesis.

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells.

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function. [BMB Reports 2015; 48(10): 583-588]