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Background Hepatocellular carcinoma (HCC) is the fifth most frequently diagnosed malignancy and the third leading cause of cancer death globally. T cells are significantly correlated with the progression, therapy and prognosis of cancer. Limited systematic studies regarding the role of T-cell-related markers in HCC have been performed. Methods T-cell markers were identified with single-cell RNA sequencing (scRNA-seq) data from the GEO database. A prognostic signature was developed with the LASSO algorithm in the TCGA cohort and verified in the GSE14520 cohort. Another three eligible immunotherapy datasets, GSE91061, PRJEB25780 and IMigor210, were used to verify the role of the risk score in the immunotherapy response. Results With 181 T-cell markers identified by scRNA-seq analysis, a 13 T-cell-related gene-based prognostic signature (TRPS) was developed for prognostic prediction, which divided HCC patients into high-risk and low-risk groups according to overall survival, with AUCs of 1 year, 3 years, and 5 years of 0.807, 0.752, and 0.708, respectively. TRPS had the highest C-index compared with the other 10 established prognostic signatures, suggesting a better performance of TRPS in predicting the prognosis of HCC. More importantly, the TRPS risk score was closely correlated with the TIDE score and immunophenoscore. The high-risk score patients had a higher percentage of SD/PD, and CR/PR occurred more frequently in patients with low TRPS-related risk scores in the IMigor210, PRJEB25780 and GSE91061 cohorts. We also constructed a nomogram based on the TRPS, which had high potential for clinical application. Conclusion Our study proposed a novel TRPS for HCC patients, and the TRPS could effectively indicate the prognosis of HCC. It also served as a predictor for immunotherapy.

In recent years, artificial intelligence technology has developed rapidly and has been gradually applied to the fields of clinical image data processing, auxiliary diagnosis and prognosis evaluation. Research has shown that it can simplify doctors’ clinical tasks, quickly provide analysis and processing results, and has high accuracy. In terms of orthodontic diagnosis and treatment, artificial intelligence can assist in the rapid fixation of two-dimensional and three-dimensional cephalometric measurements. In addition, it is also widely used in the efficient processing and analysis of three-dimensional dental molds data, and shows considerable advantages in determining deciding whether orthodontic treatment needs tooth extraction, thus assisting in judging the stage of growth and development, orthodontic prognosis and aesthetic evaluation. Although the application of artificial intelligence technology is limited by the quantity and quality of training data, combining it with orthodontic clinical diagnosis and treatment can provide faster and more effective analysis and diagnosis and support more accurate diagnosis and treatment decisions. This paper reviews the current application of artificial intelligence technology in orthodontic diagnosis and treatment in the hope that orthodontists can rationally treat and use artificial intelligence technology in the clinic, and make artificial intelligence better serve orthodontic clinical diagnosis and treatment, so as to promote the further development of intelligent orthodontic diagnosis and treatment processes.

Objectives To develop a coupled-lines system to determine the anteroposterior position of maxillary central incisors (U1) for smiling profile esthetics. Materials and Methods Thirty Asian females with ordinary and good facial harmony were selected as the study sample and the control sample, respectively. Three-dimensional facial images and 45°- and 90°-angled profiles were collected. The anteroposterior relationships between U1 and upper- and mid-facial soft tissue landmarks were measured. By morphing photos of the study sample, two artificial images were created to represent the well-balanced 45°- and 90°-angled profiles and were further processed with combined variations of soft tissue subnasale (SSn)–Glabella and the mid-point of facial axial points of the bilateral central incisor (mFA)–SSn distances. Esthetic assessments were performed on these images by layperson (n = 94) and orthodontist (n = 94) raters. Results Both upper- and mid-facial soft tissue landmarks were indispensable in assessing anteroposterior positions of U1 for well-balanced smiling profiles. As assessed in 45°- and 90°-angled profiles, the most esthetically sensitive parameters were mFA-Glabella and mFA-SSn distances. A coupled-lines system was constructed, comprising the Glabella and SSn vertical lines. In smiling profiles with optimal esthetics, the mFA point was at 2 to 5 mm posterior to the Glabella vertical and concomitantly 4 to 7 mm posterior to the SSn vertical, as perceived by orthodontists. Laypersons gave a wider range for mFA-Glabella distances, at 2 to 6 mm. Conclusions The coupled-lines system could serve as a reliable reference for determining esthetically optimal anteroposterior positions of U1 for female facial profiles.

Additional file 1. P-values for paired comparison of the deviations.

Additional file 2. 95% confidence intervals (CIs) of the means or medians of the deviations of the mandibular dentition.

Objectives: This study aimed to investigate the application effect of internet technology for managing patients with hypertension in a medical center. Methods: From December 2017 to December 2018, 400 patients with hypertension in two community health service stations affiliated to Beijing Shijitan Hospital (China) were enrolled in the present study. According to the alternating method, these patients were divided into a control group (routine medical hypertension management) and an e-intervention group (internet technical secondary hypertension management). Blood pressure levels after intervention in both groups, as well as blood pressure control, medication compliance, active health behaviors, and two-way referral rate were observed. Results: Following the interventions, blood pressure levels were significantly lower in the e-intervention group compared with the control group, and the proportion of good blood pressure control, medication compliance, active health behavior, and two-way referral were all significantly higher compared with the control group. Conclusion: The application of internet technology in medical associations may help to decrease patients’ blood pressure and improve the effective management of middle-aged and older patients with hypertension in communities.

Microgravity impacts various aspects of human health. Yet the mechanisms of spaceflight-induced health problems are not elucidated. Here, we mapped the fusion systemic analysis of the serum metabolome and the circulating microRNAome in a hindlimb unloading rat model to simulate microgravity. The response of serum metabolites and microRNAs to simulated microgravity was striking. Integrated pathway analysis of altered serum metabolites and target genes of the significantly altered circulating miRNAs with Integrated Molecular Pathway-Level Analysis (IMPaLA) software was mainly suggestive of modulation of neurofunctional signaling pathways. Particularly, we revealed significantly increased miR-383-5p and decreased aquaporin 4 (AQP4) in the hippocampus. Using rabies virus glycoprotein-modified exosomes, delivery of miR-383-5p inhibited the expression of AQP4 not only in rat C6 glioma cells in vitro but also in the hippocampus in vivo. Using bioinformatics to map the crosstalk between the circulating metabolome and miRNAome could offer opportunities to understand complex biological systems under microgravity. Our present results suggested that the change of miR-383-5p level and its regulation of target gene AQP4 was one of the potential molecular mechanisms of microgravity-induced cognitive impairment in the hippocampus.

Space bone loss is the most serious medical problem experienced by astronauts during mid- and long-term flights. Previous reports had shown that miRNAs were associated in bone remodeling and space bone loss. The present study is addressed screening and identify novel miRNAs associated with the skeletal response to gravity changes which play important roles in the regulation of bone formation. Tail-suspension and cell clinorotation were used to simulate microgravity. sRNA deep sequencing, bioinformatics analysis and clone technique were performed to find potential novel gravity-response miRNAs in the bone femur after 28 days’ rat tail-suspension. Six candidate novel miRNAs were selected and analyzed their expression in simulated microgravity and fluid shear stress conditions by quantitative real-time polymerase chain reaction (RT-qPCR). The expression of five novel miRNAs (RN4, RN21, RN43, RN48 and RN50) were changed in tail-suspended rat femurs and clinorotated UMR-106 cells, and up-regulated in UMR-106 after fluid force stress stimulation. Furthermore, The prediction and their enrichment analysis of miRNA targets showed the potential pathways by which RN4, RN43 and RN50 affected osteoblast differentiation and proliferation. Subsequently, we performed and identified the effects of RN4, RN43 and RN50 on the differentiation and proliferation of UMR-106. Taken together, we identified 3 novel miRNAs in bone which may respond to gravity change and regulate osteoblast differentiation or proliferation.

Supplemental Digital Content is available in the text, Background: Numerous treatment modalities have been attempted for masticatory muscle pain in patients with temporomandibular disorders (TMD). To compare the treatment efficacy of more than 2 competing treatments, a network meta-analysis (NMA) was conducted. Methods: This study was reported with reference to the extended Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement for reporting of systematic reviews incorporating network meta-analyses. Medline via Pubmed, Embase via OVID, and Cochrane Library Central were searched (up to February 11, 2019). Axis I protocol of Diagnostic Criteria or Research Diagnostic Criteria for Temporomandibular Disorders (DC/TMD, RDC/TMD) were chosen as diagnostic standards. The PICOS (Problem/patient, Intervention, Comparison, Outcome, Study design) method was used to screen trials under eligibility criteria. And the NMA was performed with mvmeta commands in Stata (StataCorp, Tex). Results: Of 766 studies searched, 12 randomized clinical trials (RCTs) were finally included. Nineteen different therapies were found and further categorized into 9 treatment modalities. The general heterogeneity was not found among included trials. But predictive intervals (PrIs) were conspicuously wider than confidential intervals (CIs) of all pairwise comparisons, indicating that heterogeneity may exist between studies. Complementary therapy showed the greatest probability (42.7%) to be the best intervention. It also had the highest mean rank (2.3) in the rankogram and the biggest value of surface under the cumulative ranking (SUCRA, 84.1%). Conclusions: Based on the limited evidence of available trials, complementary therapy seemed to be slightly more effective than remaining treatment modalities for pain reduction in TMD patients with masticatory muscle pain. High-quality randomized controlled trials are expected to validate the findings.

Osteocalcin (OCN) was the most abundant noncollagen protein and considered as an endocrine factor. However, the functions of Undercarboxylated osteocalcin (ucOCN) on osteoclast and bone resorption are not well understood. In the present study, preosteoclast RAW264.7 cells and bone marrow mononuclear cells (BMMs) were treated with ucOCN purified from prokaryotic bacteria. Our results showed that ucOCN attenuated the proliferation of RAW264.7 cells with a concentration dependant manner by MTS assay. Scrape wounding assay revealed the decreased motility of RAW264.7 cells after ucOCN treatment. RT-qPCR results manifested the inhibitory effects of ucOCN on the expression of osteoclastic marker genes in RAW264.7 cells during inducing differentiation of RANKL. It was also observed that ucOCN inhibited the formation of multinucleated cells from RAW264.7 cells and BMMs detected by TRAP staining. The number and area of bone resorb pits were also decreased after treatment with ucOCN during their osteoclast induction by toluidine blue staining. The formation and integrity of the osteoclast actin ring were impaired by ucOCN by immunofluorescent staining. Time dependant treatment of ucOCN during osteoclastic induction demonstrated the inhibitory effects mainly occurred at the early stage of osteoclastogenesis. Signaling analysis of luciferase activity of the CRE or SRE reporter and ERK1/2 phosphorylation showed the selective inhibitor or siRNA of Gprc6a (a presumptive ucOCN receptor) could attenuate the promotion of ucOCN on CRE-luciferase activity. Taken together, we provided the first evidence that ucOCN had negative effects on the early differentiation and bone resorption of osteoclasts via Gprc6a.

The medical assistance to advanced schistosomiasis patients established by the Chinese government is a major public facility for patients with advanced schistosomiasis. Since the medical assistance to advance schistosomiasis patients in Hunan Province started ten years ago, a set of mature and operable programs with whole program management and related technologies has been developed. The author investigated the data on medical assistance to advanced schistosomiasis patients in Hunan Province during the last 10 years (from 2006 to 2015) retrospectively, and found that the program had high therapeutic effect and high satisfaction degree of both patients and the society. In order to improve the management of the medical assistance to advanced schistosomiasis patients and share our experiences of the whole program management and related technologies with the colleagues of other provinces, this paper mainly illustrates the experiences of the program, as well as the existing problems and related strategies.

This paper summarizes the clinical diagnosis and treatment of imported African schistosomiasis, in order to make the therapeutic standards. Imported African schistosomiasis includes mainly schistosomiasis haematobia and schistosomiasis mansoni in China. In order to set up the operational standards, enhance diagnostic and cure rates, and reduce the complications, we review the related literature combined with our experience over years, and summarize, in this paper, the pathogenic mechanism, and key points of clinical diagnosis and treatment of schistosomiasis haematobia and schistosomiasis mansoni, so as to provide the reference for clinical doctors.

Fibrous dysplasia (FD) is a disease caused by postzygotic activating mutations of GNAS (R201C and R201H) that encode the α-subunit of the Gs stimulatory protein. FD is characterized by the development of areas of abnormal fibroosseous tissue in the bones, resulting in skeletal deformities, fractures, and pain. Despite the well-defined genetic alterations underlying FD, whether GNAS activation is sufficient for FD initiation and the molecular and cellular consequences of GNAS mutations remains largely unresolved, and there are no currently available targeted therapeutic options for FD. Here, we have developed a conditional tetracycline (Tet)-inducible animal model expressing the GαsR201C in the skeletal stem cell (SSC) lineage (Tet-GαsR201C/Prrx1-Cre/LSL-rtTA-IRES-GFP mice), which develops typical FD bone lesions in both embryos and adult mice in less than 2 weeks following doxycycline (Dox) administration. Conditional GαsR201C expression promoted PKA activation and proliferation of SSCs along the osteogenic lineage but halted their differentiation to mature osteoblasts. Rather, as is seen clinically, areas of woven bone admixed with fibrous tissue were formed. GαsR201C caused the concomitant expression of receptor activator of nuclear factor kappa-B ligand (Rankl) that led to marked osteoclastogenesis and bone resorption. GαsR201C expression ablation by Dox withdrawal resulted in FD-like lesion regression, supporting the rationale for Gαs-targeted drugs to attempt FD cure. This model, which develops FD-like lesions that can form rapidly and revert on cessation of mutant Gαs expression, provides an opportunity to identify the molecular mechanism underlying FD initiation and progression and accelerate the development of new treatment options.

Integrins are mechanoreceptors and mediate mechanotransduction by transferring forces into focal adhesions. Osteoclastogenesis starts with adhesion to the bone matrix, leading to cytoskeleton reorganization that is important for osteoclast polarization and migration. However, there are several signaling pathways mediating this process, and integrins have been shown to be important signaling molecules participating osteoclastogenesis. Particularly, integrins have played an important role in both force transduction and osteoclastogenesis. This paper reviewed the findings on the relationship between integrins and reorganization of osteoclast cytoskeleton under orthodontic force.

Primary dysmenorrhea (PD) is one of the most common diseases in gynecology at present. Some clinical trials have reported the effects of moxibustion and confirmed temporal factors are the important elements influencing the efficacy of moxibustion. However, no systematic review has yet been conducted. In this study, we assessed the effects of moxibustion in patients with PD enrolled in randomized controlled trials (RCTs) and the difference among different intervention times to start moxibustion. We extracted data for studies searched from 10 electronic databases and evaluated the methodological quality of the included studies. We discussed three outcomes: effective rate, pain remission, and the level of PGF2α in serum. Current clinical researches showed that, compared with nonmoxibustion treatments for PD, moxibustion leads to higher effective rate and lower level of PGF2α in serum. However, there was no difference in using moxibustion to treat PD at different intervention times. Based on the theory of Chinese medicine and the results of this study, choosing 5 ± 2 days before menstruation to start moxibustion can achieve good efficacy for PD patients. However, more high-quality RCTs are needed to confirm the conclusions.

BACKGROUND Mucus hypersecretion in the respiratory tract and goblet cell metaplasia in the airway epithelium contribute to the morbidity and mortality associated with airway inflammatory diseases. This study aimed to examine the effect and mechanisms of simvastatin on airway mucus hypersecretion in rats treated with lipopolysaccharide (LPS). METHODS Mucus hypersecretion in rat airways was induced by intra-tracheal instillation of LPS. Rats treated with or without LPS were administered intra-peritoneally simvastatin (5 and 20 mg/kg) for 4 days. Expression of Muc5ac, RhoA and mitogen-activated protein kinases (MAPK) p38 in lung were detected by real-time polymerase chain reaction (PCR), immunohistochemistry or Western blotting. Tumor necrosis factor (TNF)-alpha and IL-8 in bronchoalveolar lavage fluid (BALF) were assayed by an enzyme-linked lectin assay and enzyme linked immunosorbent assay (ELISA). RESULTS Simvastatin attenuated LPS-induced goblet cell hyperplasia in bronchial epithelium and Muc5ac hypersecretion at both the gene and protein levels in lung (P

Lack of gravity during spaceflight has profound effects on cardiovascular system, but little is known about how the cardiomyocytes respond to microgravity. In the present study, the effects of spaceflight on the structure and function of cultured cardiomyocytes were reported. The primary cultures of neonatal rat cardiomyocytes were carried on Shenzhou-6 spacecraft and activated at 4 h in orbit. 8 samples were fixed respectively at 4, 48 and 96 h after launching for immunofluorescence of cytoskeleton, and 2 samples remained unfixed to analyze contractile and secretory functions of the cultures. Ground samples were treated in our laboratory in parallel. After 115 h spaceflight, video recordings displayed that the number of spontaneous beating sites in flown samples decreased significantly, and the cells in the beating aggregate contracted in fast frequency without synchrony. Radioimmunoassay of the medium showed that the atrial natriuretic peptide secreted from flown cells reduced by 59.6%. Confocal images demonstrated the time-dependant disassembly of mirotubules versus unchanged distribution and organization of microfilaments. In conclusion, above results indicate reduced function and disorganized cytoskeleton of cardiomyocytes in spaceflight, which might provide some cellular basis for further investigations to probe into the mechanisms underlying space cardiovascular dysfunction.

OBJECTIVE: To investigate the functions of human periodontal myofibroblast (MFB) in vitro. METHODS: Human periodontal fibroblast (hPDLFs) was cultured and induced to MFB by transforming growth factor-β1 (TGF-β1). MFB was denoted as the experimental group, whereas the hPDLFs was the control group. The groups were continuously cultured and harvested at 0, 12, 24, 48, and 72 h. The MFB marker α-smooth muscle actin (α-SMA) was examined by immunocytochemistry. The expression of fibronectin (FN) between MFB was examined by immunocytochemistry to detect the MFB contact relationship. The mRNA expression levels of α-SMA, collagen (Col )Ⅰ, and Col Ⅲ were measured by reverse transcription-polymerase chain reaction (RT-PCT) to analyze extracellular matrix secretion. The protein expression levels of α-SMA and Col Ⅰ were also assessed by Western blot. RESULTS: The experimental group had significantly higher α-SMA expression than the control group at 0 h (P

Introduction Microgravity is thought to decrease osteoblastic activity and induce osteoporosis during spaceflight, but the mechanisms, particularly the attendant changes in gene expression, are not well understood. It is suspected that the cytoskeletal system is involved in the manifold changes of cell shape, function, and signaling under microgravity conditions. Methods We constructed cell lines stably transfected with pJI36EGFP and pJI23EGFP, which contained a 3.6 and a 2.3 kb fragment, respectively, of the α1(I) collagen gene (COL1A1) promoter fused with the enhanced green fluorescence protein (EGFP) reporter gene. We then developed a semi-quantitative analysis of EGFP fluorescence intensity to evaluate the effects of clinorotation and/or cytochalasin B on the activity of the COL1A1 promoter. Simultaneously, we assessed the collagen type I protein content versus total protein content in clinorotated or control osteoblasts, using immunocytochemistry and the Bradford method, respectively. Results The fluorescence intensity analysis revealed that the expression of COL1A1-EGFP increased in GFP-ROS cells clinorotated for 24 or 48 h, as compared with stationary control cultures. We observed a similar trend in collagen type I content, as assessed by immunocytochemistry. We found that the osteoblast microfilaments tended to disassemble and show a reduction in stress fibers under space flight and clinorotation. Treatment with cytochalasin B in normal gravity resulted in a dose-dependent increase of EGFP fluorescence intensity, indicating that disruption of the actin system was associated with increased activity of the COL1A1 promoter. Conclusion Our study demonstrates that disrupting the actin cytoskeleton by treatment with cytochalasin B and real or simulated microgravity conditions led to altered COL1A1 promoter activity. Together, these results suggest that actin may participate in the regulation of the COL1A1 promoter activity under microgravity conditions.

Arylalkylamine N-acetyltransferase (AA-NAT) is critical enzyme in Melatonin (MLT) biosynthesis for MLT regulating the animal seasonal breeding. In this study, DNA sequencing methods were applied to detect the polymorphisms of the AA-NAT gene in 179 Chinese sheep belonging to two non-seasonal reproduction breeds and two seasonal reproduction breeds. One mutation at exon 3 (NM_001009461:c.486A > G) was firstly described at the sheep AA-NAT locus. Hence, we described the Sma I PCR-RFLP method for detecting EX3 486A > G mutation, frequencies of the AA-NAT-G allele varied from 0.871 to 0.908 in two non-seasonal reproduction breeds and 0.517 to 0.578 in two seasonal reproduction breeds. The associations of Sma I polymorphism with estrus traits was analyzed in non-seasonal reproduction breeds sheep and seasonal reproduction breeds sheep, the significant statistical results were found between them, the GG genotype frequencies was higher in non-seasonal reproduction breeds (p G mutation could facilitate association analysis and serve as a genetic marker for Chinese sheep breeding and genetics.

Firstly, it is briefly introduced that Guangxi characteristic economic agricultural products are currently characterized by typical regional features and strong competitiveness. Then, Guilin is taken as an example, the problems existing in constructing the supply chain and brand of Guilin characteristic economic agricultural products are pointed out after the analysis of its current situations, the problems include weak agricultural production infrastructure, weak deep products processing capacity, low modernization level of storage and transportation as well as short of fixed marketing channels; meanwhile, brand construction faces the problems of little top brands and few differentiated products. Finally, some measures are proposed to solve these problems from the perspectives of establishing the management idea of agricultural products supply chain and enhancing the awareness to protect agricultural products brand, it is also suggested that these measures be extended to the whole Guangxi autonomous region in order to explore the way of the development of Guangxi characteristic agriculture.

Firstly, it is briefly introduced that Guangxi characteristic economic agricultural products are currently characterized by typical regional features and strong competitiveness. Then, Guilin is taken as an example, the problems existing in constructing the supply chain and brand of Guilin characteristic economic agricultural products are pointed out after the analysis of its current situations, the problems include weak agricultural production infrastructure, weak deep products processing capacity, low modernization level of storage and transportation as well as short of fixed marketing channels; meanwhile, brand construction faces the problems of little top brands and few differentiated products. Finally, some measures are proposed to solve these problems from the perspectives of establishing the management idea of agricultural products supply chain and enhancing the awareness to protect agricultural products brand, it is also suggested that these measures be extended to the whole Guangxi autonomous region in order to explore the way of the development of Guangxi characteristic agriculture.

A novel VECA (vascular endothelial cell antigen) was previously identified by using an antibody pool against antigens in HUVECs (human umbilical-vein endothelial cells). VECA has been evolutionarily conserved in vertebrate species ranging from frog and fish to mouse and human. Bioinformatics analysis indicated that VECA was 10.1 kDa in size, with a predicted signal sequence and transmembrane domain, indicating that VECA may have important biological functions. The present paper describes a procedure for obtaining and purifying human recombinant VECA expressed in Escherichia coli as a fusion protein, via a human VECA cDNA linked pQE30 expression vector to DNA coding for hexahistidine. The purified protein was used to raise anti-(human VECA) polyclonal antibodies, which were suitable for detecting the presence of VECA in cells, cell-culture supernatant and tissues by immunoblotting and immunohistochemistry. To our knowledge, this is the first study on the protein expression and polyclonal-antibody production for human VECA. In addition, we report for the first time the positive identification of VECA in humans at the protein and subcellular level and provide the first experimental verification that VECA was indeed a secreted protein. The anti-(human VECA) polyclonal antibodies prepared may serve as a useful tool for future biological function studies on VECA.

It is well known that cytoskeleton system is the sensor of gravity in cells. Under microgravity condition, cytoskeleton is associated with the changes of cell shape, function, signaling and so on; but the relationship between cytoskeleton and gene expression is not fully understood. In present study, we discussed the effects of cell microfilament on the activity of collagen type I alpha 1 chain gene (COL1A1) promoter under microgravity simulated by clinostat and/or cytochalasin B as microfilament depolymerizer in the established EGFP-ROS cell line using the method of fluorescence semi-quantitative analysis and the fluorescent stain of microfilament. Compared with the normal control, the microfilament of ROS17/2.8 cell tended to disassemble, marginal distribution of fiber stress, and showed reducing stress fibers after spaceflight in Photon-M1 or clinorotation simulated microgravity, which suggested that microgravity destroyed the well-order cell cytoskeleton and induced a rearrangement. Treatment with suitable concentration of cytochalasin B in normal gravity induced disruption of microfilament, increased the activity of COL1A1 promoter and resulted in a dose-dependent increase of EGFP fluorescence. Therefore, a certain extent disruption of the microfilament system was associated with increased activity of the COL1A1 promoter. All above demonstrate that microfilament cytoskeleton system takes part in the regulation of COL1A1 promoter activity and plays an important role in the signaling of microgravity.

To obtain ROS17/2.8 cell lines which were stably expressing EGFP reporter gene drived by COL1A1 promoter.A 3.6 Kb COL1A1 promoter from rat was cloned into pMD-18-T vector by PCR. This amplified promoter vector was digested to get several different length fragments which were then fused with EGFP reporter gene to construct eukaryotic expression vectors. ROS17/2.8 cell was stably transfected with these vectors by LipofectAMINE(TM) and selected by G418.The COL1A1-EGFP stably transfected cell lines were established.The cell lines will be useful for studying the effects of microgravity on the activity of COL1A1 promoter and expression of gene related with bone form.

Bone-formation related gene plays a critical role in bone loss induced by space microgravity, however the exact mechanism is unclear. In this study, we aim to investigate the effect of microgravity on the activity of alpha 1(I) collagen (COL1A1) gene promoter and the expression of osteoblast-related genes. COL1A1 promoter was digested by restriction enzymes resulting in three DNA fragments. The fragments were ligated with the enhanced green fluorescent protein report gene, and subcloned into expression vectors. ROS17/2.8 cells transfected by these vectors were screened by G418, and enhanced green fluorescent protein (EGFP) positive colonies were isolated and cultured under clinostat condition. EGFP and Collagen type I expression level were detected by fluorescence intensity analysis and immunocytochemistry methods respectively. The results showed that the expression of EGFP and collagen type I was increased 24 h, 48 h after the cells were cultured under stimulated microgravity, illustrating that the activity of COL1A1 promoter might be increased. In conclusion, osteoblasts can compensatively increase the expression of type I collagen by enhancing the activity of COL1A1 promoter under short-term simulated microgravity conditions.

Microgravity decreases osteoblastic activity, induces actin microfilament disruption and inhibits the responsiveness of osteoblast to cytokines, but the mechanisms remains enigmatic. The F-actin cytoskeleton has previously been implicated in manifold changes of cell shape, function and signaling observed under microgravity. Here we investigate the involvement of microfilament in mediating the effects of microgravity and BMP2 induction on Cbfa1 activity. For this purpose we constructed a fluorescent reporter cell line (OSE-MG63) of Cbfa1 activity by stably transfecting MG63 cells with a reporter consisting of six tandem copies of OSE2 and a minimal mOG2 promoter upstream of enhanced green fluorescent protein (EGFP). The fluorescence intensity of OSE-MG63 showed responsiveness to bone-related cytokines (IGF-I, vitamin D3 and BMP2) and presented an accordant tendency with alkaline phosphatase (ALP) activity. Using OSE-MG63 reporter fluorescence, we performed a semi-quantitative analysis of Cbfa1 activity after treatment with simulated microgravity, microfilament-disrupting agent (cytochalasin B, CB), microfilament-stabilizing agent (Jasplakinolide, JAS) or any combination thereof. In parallel, ALP activity, DNA binding activity of Cbfa1 to OSE2 (ChIP), F-actin structure (immunofluorescence) and EGFP mRNA expression (RT-qPCR) were analyzed. Simulated microgravity inhibited Cbfa1 activity, affected the responsiveness of Cbfa1 to cytokine BMP2, and caused a thinning and dispersed distribution of microfilament. Under normal gravity, CB significantly attenuated BMP2 induction to Cbfa1 activity as well as DNA binding activity of Cbfa1 to OSE2. The addition of JAS reversed the inhibitory effects of microgravity on the responsiveness of Cbfa1 to BMP2. Our study demonstrates that disrupting the microfilament organization by CB or simulated microgravity attenuates the responsiveness of Cbfa1 to BMP2. A stabilization of the microfilament organization by JAS reverses this inhibition. Taken together, these results suggest that actin microfilament participates in BMP2's induction to Cbfa1 activity and that their disruption might be an important contributor to microgravity's inhibition on BMP2's osteogenic induction.