MiR-449 overexpression inhibits osteogenic differentiation of bone marrow mesenchymal stem cells via suppressing Sirt1/Fra-1 pathway in high glucose and free fatty acids microenvironment.

Diabetic osteoporosis is a chronic complication caused by diabetes mellitus, and However, the exact mechanism of diabetes mellitus-induced osteoporosis is still unknown. In this study, we investigate the effect of miR-449 on osteogenic differentiation and its underlying mechanism in human bone marrow-derived mesenchymal stem cells (hBMSCs) with high glucose (HG) and free fatty acids (FFA) treatment. Results showed that after culturing for 14 days, high glucose (HG) and free fatty acids (FFA) treatment dramatically decreased mineralization of human bone marrow-derived mesenchymal stem cells (hBMSCs) compared with cells treated with osteogenic medium (OM) alone. We also found that miR-449 expression was up-regulated during osteogenic differentiation of hBMSCs with HG and FFA treatment. Moreover, during osteogenic differentiation of hBMSCs with HG and FFA treatment, miR-449 mimics notably decreased the alkaline phosphatase (ALP) activity and the mRNA and protein expression levels of runt-related transcription factor 2 (Runx2), ALP, collagen I, osteocalcin (OCN), and bone sialoprotein (BSP), which was remarkably increased by miR-449 inhibitors. Furthermore, miR-449 directly targets Sirt1 by binding to its 3'-UTR. Sirt1 overexpression reverses the suppressive effect of miR-449 mimics on Fra-1 mRNA and protein expression, which was also alleviated by Fra-1 overexpression. In addition, Fra-1 overexpression alleviates the inhibitory effect of miR-449 mimics on the ALP activity and the mRNA and protein of Runx2, collagen I, OCN and BSP. Taken together, our results indicated that miR-449 overexpression inhibited osteogenic differentiation of HG-FFA-treated hBMSCs through the Sirt1/Fra-1 signal pathway. It is conceivable that modulating miR-449 might provide a new therapy for intervention in diabetic osteoporosis.

An Improved Method for Predicting Linear B-cell Epitope Using Deep Maxout Networks.

To establish a relation between an protein amino acid sequence and its tendencies to generate antibody response, and to investigate an improved in silico method for linear B-cell epitope (LBE) prediction. We present a sequence-based LBE predictor developed using deep maxout network (DMN) with dropout training techniques. A graphics processing unit (GPU) was used to reduce the training time of the model. A 10-fold cross-validation test on a large, non-redundant and experimentally verified dataset (Lbtope_Fixed_ non_redundant) was performed to evaluate the performance. DMN-LBE achieved an accuracy of 68.33% and an area under the receiver operating characteristic curve (AUC) of 0.743, outperforming other prediction methods in the field. A web server, DMN-LBE, of the improved prediction model has been provided for public free use. We anticipate that DMN-LBE will be beneficial to vaccine development, antibody production, disease diagnosis, and therapy.

Reactive oxygen species induce cell death via Akt signaling in rat osteoblast-like cell line ROS 17/2.8.

In bones, osteoblasts are responsible for bone formation. The cell death of osteoblasts may cause a series of bone diseases and lead to bone loss, such as osteoarthrosis, hyperparathyroidism, and Paget's disease. Reactive oxygen species (ROS) are reported as a main factor for osteoblast cell death and further several bone diseases. However, the detailed mechanism is still largely unknown. Here, we found that ROS could induce cell death of rat osteoblast-like cell line ROS 17/2.8 via Akt (protein kinase B). Also, the mammalian target of rapamycin signaling was involved in this process. Our findings could help to reveal the cellular mechanism of osteoblast cell death, which is served for the pursuit of clinical treatment targets of relative bone diseases.

EPMLR: sequence-based linear B-cell epitope prediction method using multiple linear regression.

BACKGROUND: B-cell epitopes have been studied extensively due to their immunological applications, such as peptide-based vaccine development, antibody production, and disease diagnosis and therapy. Despite several decades of research, the accurate prediction of linear B-cell epitopes has remained a challenging task. RESULTS: In this work, based on the antigen's primary sequence information, a novel linear B-cell epitope prediction model was developed using the multiple linear regression (MLR). A 10-fold cross-validation test on a large non-redundant dataset was performed to evaluate the performance of our model. To alleviate the problem caused by the noise of negative dataset, 300 experiments utilizing 300 sub-datasets were performed. We achieved overall sensitivity of 81.8%, precision of 64.1% and area under the receiver operating characteristic curve (AUC) of 0.728. CONCLUSIONS: We have presented a reliable method for the identification of linear B cell epitope using antigen's primary sequence information. Moreover, a web server EPMLR has been developed for linear B-cell epitope prediction: http://www.bioinfo.tsinghua.edu.cn/epitope/EPMLR/ .

PDGF-regulated miRNA-138 inhibits the osteogenic differentiation of mesenchymal stem cells.

Differentiation-specific microRNAs may play a critical role in MSC differentiation, and they can be altered by PDGF signaling. We propose that PDGF modulates MSC differentiation by regulating microRNA expression. Therefore, we investigated whether PDGF treatment could alter the expression profile of miRNAs in MSCs. Furthermore, we assessed the osteoblast phenotype of MSCs after inducing osteogenic differentiation. We found that PDGF treatment significantly inhibits the osteogenic differentiation of MSCs and that miR-138 gene transcription is controlled by PDGF signaling. Our results confirm that miR-138 inhibits the osteogenic differentiation of MSCs and suppresses the phosphorylation of FAK, ERK1/2, and Runx2. Furthermore, our study clearly demonstrates that downregulation of Runx2 by miR-138 is critical for the PDGF-mediated inhibition of osteogenic differentiation of MSCs. These findings indicate that inhibition of miR-138 function in MSCs, either by treatment with anti-miR-138 or by overexpression of the miR-138 target sequence (miRNA sponge), could represent a potential therapeutic strategy for the treatment of bone homeostasis disorders caused by activation of the PDGF pathway.

Comparison of 2 kinds of pedicle screws in primary spinal instrumentation: biomechanical and interfacial evaluations in sheep vertebrae in vitro.

STUDY DESIGN: Expansive pedicle screw (EPS) and polymethylmethacrylate-augmented pedicle screw (PMMA-PS) were inserted in sheep vertebrae in vitro and were evaluated by performing biomechanical tests, radiographic examinations and histological observations. OBJECTIVE: The objective of the study was to compare the biomechanical and interfacial performances of EPS and PMMA-PS in sheep lumbar vertebrae in vitro. SUMMARY OF BACKGROUND DATA: It is a great challenge for orthopedic surgeons performing transpedicular fixation in the osteoporotic spine. It was reported that either the EPS or PMMA-PS could increase the screw stability. However, there are no studies comparing the 2 kinds of screws especially in primary spinal instrumentation. METHODS: A total of 60 sheep lumbar vertebrae were randomly divided into 3 groups. A pilot hole was made in advance in all samples using the same method. Thereafter, the conventional pedicle screw (CPS) was inserted directly into the pilot hole in the CPS group; the hole in PMMA-PS group was first filled with polymethylmethacrylate (PMMA; 1.0 mL) and then inserted with CPS; and the EPS was inserted directly into the vertebrae in EPS group. After a period of 24 hours, biomechanical tests were performed to evaluate screw stability, and x-ray examination, micro-computerized tomography analysis, and histologic observation were performed to evaluate the interface between screw and bone. RESULTS: Compared with the stability of CPS, those of EPS and PMMA-PS were significantly enhanced. However, no significant differences were detected between the stabilities of EPS and PMMA-PS. The PMMA surrounding the screw blocked direct contact between bone and screw and formed a "screw-PMMA-bone" interface in the PMMA-PS group. There was a "screw-bone" interface in both CPS and EPS groups. Nevertheless, the expanded anterior part of EPS formed a claw-like structure pressing the surrounding bone trabeculae, which made the local bone tissue more compacted and denser than that in the CPS group. CONCLUSIONS: EPS can enhance the screw stability as markedly as the traditional PMMA-PS in primary surgery, and EPS can form a better immediate interface between screw and bone compared with PMMA-PS. EPS also can effectively avoid thermal injury, leakage, and compression caused by PMMA. A great feasibility was proved in this study to perform comparisons between the 2 kinds of pedicle screws in osteoporotic sheep vertebrae in vivo in the further research. In conclusion, we propose that EPS has a great application potential in augmentation of screw stability in the clinic.

A sequence-based evolutionary distance method for Phylogenetic analysis of highly divergent proteins.

Because of the limited effectiveness of prevailing phylogenetic methods when applied to highly divergent protein sequences, the phylogenetic analysis problem remains challenging. Here, we propose a sequence-based evolutionary distance algorithm termed sequence distance (SD), which innovatively incorporates site-to-site correlation within protein sequences into the distance estimation. In protein superfamilies, SD can effectively distinguish evolutionary relationships both within and between protein families, producing phylogenetic trees that closely align with those based on structural information, even with sequence identity less than 20%. SD is highly correlated with the similarity of the protein structure, and can calculate evolutionary distances for thousands of protein pairs within seconds using a single CPU, which is significantly faster than most protein structure prediction methods that demand high computational resources and long run times. The development of SD will significantly advance phylogenetics, providing researchers with a more accurate and reliable tool for exploring evolutionary relationships.

Biomechanical comparison of different techniques in primary spinal surgery in osteoporotic cadaveric lumbar vertebrae: expansive pedicle screw versus polymethylmethacrylate-augmented pedicle screw.

INTRODUCTION: Transpedicular fixation can be challenging in the osteoporotic spine. Expansive pedicle screw (EPS) and polymethylmethacrylate-augmented pedicle screw (PMMA-PS) were both used to increase screw stability. However, there are a little or no biomechanical comparisons of EPS and PMMA-PS, especially in primary spinal surgery in osteoporotic vertebrae. The purpose of this study was to compare the stability of EPS and PMMA-PS in primary spinal surgery. MATERIALS AND METHODS: Fifteen osteoporotic vertebrae were randomly divided into three groups. The conventional pedicle screw (CPS) was inserted in CPS group, the pilot hole was filled with PMMA followed by CPS insertion in PMMA-PS group, and EPS was inserted in EPS group. Twenty-four hours later, X-ray and CT examination and biomechanical tests were performed to all vertebrae. RESULTS: In PMMA-PS group, PMMA existed in bone tissue around the CPS in both vertebral body and pedicle of vertebral arch, and PMMA surrounding the screw formed a spindle-shaped structure in vertebral body. In EPS group, anterior part of EPS presented an obvious expansion in vertebral body and formed a clawlike structure. Screw stabilities in PMMA-PS and EPS groups were significantly enhanced compared with those in CPS group (P < 0.05). However, there was no significant difference between PMMA-PS and EPS groups (P > 0.05). CONCLUSION: Expansive pedicle screw can markedly enhance screw stability with a similar effect to the traditional method of screw augmentation with PMMA in primary surgery in osteoporotic vertebrae. In addition, EPS can overcome pedicle fracture, leakage and compression caused by lager screw and augmentation with PMMA. We propose that EPS is an effective, safe and easy method and has a great application potential in augmentation of screw stability in osteoporosis in clinic.

Thermal unfolding of Escherichia coli trigger factor studied by ultra-sensitive differential scanning calorimetry.

Temperature-induced unfolding of Escherichia coli trigger factor (TF) and its domain truncation mutants, NM and MC, were studied by ultra-sensitive differential scanning calorimetry (UC-DSC). Detailed thermodynamic analysis showed that thermal induced unfolding of TF and MC involves population of dimeric intermediates. In contrast, the thermal unfolding of the NM mutant involves population of only monomeric states. Covalent cross-linking experiments confirmed the presence of dimeric intermediates during thermal unfolding of TF and MC. These data not only suggest that the dimeric form of TF is extremely resistant to thermal unfolding, but also provide further evidence that the C-terminal domain of TF plays a vital role in forming and stabilizing the dimeric structure of the TF molecule. Since TF is the first molecular chaperone that nascent polypeptides encounter in eubacteria, the stable dimeric intermediates of TF populated during thermal denaturation might be important in responding to stress damage to the cell, such as heat shock.

The contribution of proline residues to protein stability is associated with isomerization equilibrium in both unfolded and folded states.

It has long been understood that the proline residue has lower configurational entropy than any other amino acid residue due to pyrrolidine ring hindrance. The peptide bond between proline and its preceding amino acid (Xaa-Pro) typically exists as a mixture of cis- and trans-isomers in the unfolded protein. Cis-trans isomerization of Xaa-Pro peptide bonds are infrequent, but still occur in folded proteins. Therefore, the effects of the cis-trans isomerization equilibrium in both unfolded and folded states should be taken into account when estimating the stability contribution of a specific proline residue. In order to study the stability contribution of the four proline residues to the hyperthermophilic protein Ssh10b, in this work, we expressed and purified a series of Pro-->Ala mutants of Ssh10b, and performed correlative unfolding experiments in detail. We proposed a new unfolding model including proline isomerization. The model predicts that the contribution of a proline residue to protein stability is associated with the thermodynamic equilibrium between cis- and trans-isomers both in the unfolded and folded states, agreeing well with the experimental results.

Refolding of the hyperthermophilic protein Ssh10b involves a kinetic dimeric intermediate.

The alpha/beta-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination. The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate, agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other alpha/beta-mixed or pure beta-sheet proteins are compared.

HIV-1 tropism prediction by the XGboost and HMM methods.

Human Immunodeficiency Virus 1 (HIV-1) co-receptor usage, called tropism, is associated with disease progression towards AIDS. Furthermore, the recently developed and developing drugs against co-receptors CCR5 or CXCR4 open a new thought for HIV-1 therapy. Thus, knowledge about tropism is critical for illness diagnosis and regimen prescription. To improve tropism prediction accuracy, we developed two novel methods, the extreme gradient boosting based XGBpred and the hidden Markov model based HMMpred. Both XGBpred and HMMpred achieved higher specificities (72.56% and 72.09%) than the state-of-the-art methods Geno2pheno (61.6%) and G2p_str (68.60%) in a 10-fold cross validation test at the same sensitivity of 93.73%. Moreover, XGBpred had more outstanding performances (with AUCs 0.9483, 0.9464) than HMMpred (0.8829, 0.8774) on the Hivcopred and Newdb (created in this work) datasets containing larger proportions of hard-to-predict dual tropic samples in the X4-using tropic samples. Therefore, we recommend the use of our novel method XGBpred to predict tropism. The two methods and datasets are available via http://spg.med.tsinghua.edu.cn:23334/XGBpred/. In addition, our models identified that positions 5, 11, 13, 18, 22, 24, and 25 were correlated with HIV-1 tropism.

A novel method for high accuracy sumoylation site prediction from protein sequences.

BACKGROUND: Protein sumoylation is an essential dynamic, reversible post translational modification that plays a role in dozens of cellular activities, especially the regulation of gene expression and the maintenance of genomic stability. Currently, the complexities of sumoylation mechanism can not be perfectly solved by experimental approaches. In this regard, computational approaches might represent a promising method to direct experimental identification of sumoylation sites and shed light on the understanding of the reaction mechanism. RESULTS: Here we presented a statistical method for sumoylation site prediction. A 5-fold cross validation test over the experimentally identified sumoylation sites yielded excellent prediction performance with correlation coefficient, specificity, sensitivity and accuracy equal to 0.6364, 97.67%, 73.96% and 96.71% respectively. Additionally, the predictor performance is maintained when high level homologs are removed. CONCLUSION: By using a statistical method, we have developed a new SUMO site prediction method - SUMOpre, which has shown its great accuracy with correlation coefficient, specificity, sensitivity and accuracy.

A novel procedure for identification of the folding/unfolding patterns of dimeric proteins.

The unfolding of proteins has been widely used for investigating the thermodynamic properties of monomeric proteins but has been used infrequently for dimeric (or oligomeric) proteins, because of the inherent cooperation of denaturation and dissociation of the dimers (oligomers). Here, we introduce a thermodynamic parameter K(obs) to discriminate the diverse folding patterns of dimeric proteins. K(obs) remains constant as the protein concentration increases for the true one-step curve of unfolding pattern (A), increases and reaches a plateau for one-step curves with monomeric intermediate pattern (B), and increases steadily with no plateau for one-step curves with dimeric intermediate pattern (C).

[Soluble expression, purification of bone morphogenetic protein-2 fragment and its antibody preparation].

The bone morphogenetic protein-2 (BMP-2) fragment (BMP-2omega, 606-846bp) cDNA was amplified from total RNA of SAOS-2 cells by using RT-PCR. The PCR product was then inserted into pET-28a (+) vector for constructing the expression plasmid that would be used to transform the host cell BL21(DE3). After IPTG inducing under different conditions, this BMP-2w protein could be expressed in high level as a soluble form, and purified by chelating column (Ni-NTA). Polyclonal antibody was made by immunizing mice with using purified protein, and the antiserum titer generated was 1: 6400 that was measured by ELISA. Western blot result showed that this antibody could bind to BMP-2 protein specifically. Above research result set up the basis for studying on the treatment of osteosarcoma.

A statistical approach to the prediction of pK(a) values in proteins.

We propose a simple model for the calculation of pK(a) values of ionizable residues in proteins. It is based on the premise that the pK(a) shift of ionizable residues is linearly correlated to the interaction between a particular residue and the local environment created by the surrounding residues. Despite its simplicity, the model displays good prediction performance. Under the sixfold cross test prediction over a data set of 405 experimental pK(a) values in 73 protein chains with known structures, the root-mean-square deviation (RMSD) between the experimental and calculated pK(a) was found to be 0.77. The accuracy of this model increases with increasing size of the data set: the RMSD is 0.609 for glutamate (the largest data set with 141 sites) and approximately 1 pH unit for lysine, with a data set containing 45 sites.

The effects of NaCl concentration and pH on the stability of hyperthermophilic protein Ssh10b.

BACKGROUND: Hyperthermophiles constitute a group of microorganisms with an optimum growth temperature of between 80 degrees C and 100 degrees C. Although the molecular underpinnings of protein thermostabilization have been the focus of many theoretical and experimental efforts, the properties leading to the higher denaturation temperature of hyperthermophilic proteins are still controversial. Among the large number of factors identified as responsible for the thermostability of hyperthermophilic proteins, the electrostatic interactions are thought to be a universally important factor. RESULTS: In this study, we report the effects of pH and salt concentration on the urea-induced denaturation of the protein Ssh10b from a hyperthermophile in low ionic strength buffer. In the absence of NaCl, the unfolding DeltaG of the protein increased from about 33 kJ/mol at pH 3 to about 78 kJ/mol at pH 10. At all values of pH, the DeltaG increased with increasing NaCl concentration, indicating that salt stabilizes the protein significantly. CONCLUSION: These findings suggests that the increased number of charged residues and ion pairs in the protein Ssh10b from hyperthermophiles does not contribute to the stabilization of the folded protein, but may play a role in determining the denatured state ensemble and also in increasing the denaturation temperature.

Determinants of Thermostability in Serine Hydroxymethyltransferase Identified by Principal Component Analysis.

Protein thermostability has received growing attention in recent years. Little is known about the determinants of thermal resistance in individual protein families. However, it is known that the mechanism is family-dependent and not identical for all proteins. We present a multivariate statistical analysis to find the determinants of thermostability in one protein family, the serine hydroxymethyltransferase family. Based on principal component analysis, we identified three amino acid fragments as the potential determinants of thermostability. The correlation coefficients between all the putative fragments and the protein thermostability were significant according to multivariable linear regression. Within the fragments, four critical amino acid positions were identified, and they indicated the contributions of Leu, Val, Lys, Asp, Glu, and Phe to thermostability. Moreover, we analyzed the insertions/deletions of amino acids in the sequence, which showed that thermophilic SHMTs tend to insert or delete residues in the C-terminal domain rather than the N-terminal domain. Our study provided a promising approach to perform a preliminary search for the determinants of thermophilic proteins. It could be extended to other protein families to explore their own strategies for adapting to high temperature.

Establishing knowledge on the sequence arrangement pattern of nucleated protein folding.

The heat-tolerance mechanisms of (hyper)thermophilic proteins provide a unique opportunity to investigate the unsolved protein folding problem. In an attempt to determine whether the interval between residues in sequence might play a role in determining thermostability, we constructed a sequence interval-dependent value function to calculate the residue pair frequency. Additionally, we identified a new sequence arrangement pattern, where like-charged residues tend to be adjacently assembled, while unlike-charged residues are distributed over longer intervals, using statistical analysis of a large sequence database. This finding indicated that increasing the intervals between unlike-charged residues can increase protein thermostability, with the arrangement patterns of these charged residues serving as thermodynamically favorable nucleation points for protein folding. Additionally, we identified that the residue pairs K-E, R-E, L-V and V-V involving long sequence intervals play important roles involving increased protein thermostability. This work demonstrated a novel approach for considering sequence intervals as keys to understanding protein folding. Our findings of novel relationships between residue arrangement and protein thermostability can be used in industry and academia to aid the design of thermostable proteins.