ALBERT

Description: Publication date: Available online 7 August 2015 Source: FEBS Open Bio Author(s): Kimberley J. Anderson, Aaron P. Russell, Victoria C. Foletta The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterised. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H 2 O 2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H 2 O 2 -induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.

Description: Publication date: Available online 12 August 2015 Source: FEBS Open Bio Author(s): Yang Song, Yonghong Zhang, Haoxiang Jiang, Yanting Zhu, Lu Liu, Wei Feng, Lan Yang, Yibin Wang, Manxiang Li Activation of the Notch3 cascade is involved in the development of pulmonary arterial hypertension by stimulating the proliferation of vascular smooth muscle cells. However, the detailed molecular mechanisms underlying this effect are still unclear. The present study aims to address this issue. We demonstrated that over-expression of intracellular domain of the Notch3 receptor (NICD3) by adenovirus transfection dramatically induced proliferation of primary cultured pulmonary artery smooth muscle cells. This was accompanied with up-regulation of Hes1 protein and down-regulation of p27Kip1protein. More importantly, we observed that prior silencing of Hes1 with siRNA blocked NICD3 over-expression-induced p27Kip1 reduction and cell proliferation. The present study suggests that Hes1 lies downstream of NICD3 and particularly mediates Notch3 signaling-induced proliferation of pulmonary arterial smooth muscle cells by down-regulation of p27Kip1 expression.

Description: Publication date: Available online 11 August 2015 Source: FEBS Open Bio Author(s): Xueying Zhang, Yuanzhe Jin, Xiaohong Zhang, Mingming Lei, Yingzi Lin, Ian L. Megson, Jun Wei, Bo Yu A number of studies have reported an association between increased levels of antibodies against oxidized low-density lipoprotein (oxLDL) and cardiovascular disease, but the anti-oxLDL antibody has not been confirmed to serve as an effective biomarker for prediction of acute myocardial infarction (AMI). Apolipoprotein B 100 (ApoB 100 )-derived peptide fragments generated by proteolytic degradation and aldehyde modification are the major antigens in oxLDL, and so the present work was undertaken to detect circulating IgG for Apo-B 100 -derived peptide antigens. An in-house enzyme-linked immunosorbent assay (ELISA) was developed with eight ApoB 100 -derived peptide antigens (Ag1-Ag8) to detect circulating anti-ApoB 100 IgG levels in 267 patients with AMI and 201 control subjects. Binary logistic regression analysis revealed that circulating IgG for Ag1 was significantly higher in the patient group than the control group ( P 〈0.001) after adjustment for age, gender, smoking, hypertension, diabetes and circulating levels of cholesterol, HDL, LDL, ApoA and ApoB 100 . None of the other seven antigens detected an increase in IgG levels in AMI patients compared with control subjects. Spearman correlation analysis showed no correlation between IgG antibody for Ag1 and clinical characteristics. In conclusion, the linear peptide antigens derived from ApoB 100 may be suitable for the development of an ELISA antibody test for prediction of AMI, although further confirmation is still needed in large-scale clinical studies.

Description: Publication date: Available online 11 August 2015 Source: FEBS Open Bio Author(s): Yu-E. Hao, Dong-Fang He, Rong-Hua Yin, Hui Chen, Jian Wang, Shao-Xia Wang, Yi-Qun Zhan, Chang-Hui Ge, Chang-Yan Li, Miao Yu, Xiao-Ming Yang G protein-coupled receptor kinase interactor 2 (GIT2) is a signaling scaffold protein involved in regulation of cytoskeletal dynamics and the internalization of G protein-coupled receptors (GPCRs). The short-splice form of GIT2 is expressed in peripheral T cells and thymocytes. However, the functions of GIT2 in T cells have not yet been determined. We show that treatment with Con A in a model of polyclonal T-lymphocyte activation resulted in marked inhibitions in the intrahepatic infiltration of inflammatory cells, cytokine response and acute liver failure in Git2 -/- mice. CD4 + T cells from Git2 -/- mice showed significant impairment in proliferation, cytokine production and signal transduction upon TCR-stimulated activation. Our results suggested that GIT2 plays an important role in T-cell function in vivo and in vitro .

Description: Publication date: Available online 3 August 2015 Source: FEBS Open Bio Author(s): Johan Vande Voorde, Peter Vervaeke, Sandra Liekens, Jan Balzarini Mycoplasmas may colonize tumour tissue in patients. The cytostatic activity of gemcitabine was dramatically decreased in Mycoplasma hyorhinis -infected tumour cell cultures compared with non-infected tumour cell cultures. This mycoplasma-driven drug deamination could be prevented by exogenous administration of the cytidine deaminase (CDA) inhibitor tetrahydrouridine, but also by the natural nucleosides or by a purine nucleoside phosphorylase inhibitor. The M. hyorhinis -encoded CDA Hyor gene was cloned, expressed as a recombinant protein and purified. CDA Hyor was found to be more catalytically active than its human equivalent and efficiently deaminates (inactivates) cytosine-based anticancer drugs. CDA Hyor expression at the tumour site may result in selective drug inactivation and suboptimal therapeutic efficiency.

Description: Publication date: Available online 19 August 2015 Source: FEBS Open Bio Author(s): Ajay Srivastava Malignant pleural mesothelioma (MPM) is an aggressive form of thoracic cancer with poor prognosis. While some studies have identified the molecular alterations associated with MPM, little is known about their role in MPM. For example, fragile X mental retardation ( FMR ) gene is up-regulated in MPM but its role in MPM is unknown. Here, utilizing Drosophila genetics, I investigate the possible role FMR may be playing in MPM. I provide evidence which suggests that FMR may contribute to tumorigenesis by up-regulating a matrix metalloprotease (MMP) and by degrading the basement membrane (BM), both important for tumor metastasis. I also demonstrate a novel link between FMR and the JNK pathway and suggest that the effects of FMR in MPM could in part be mediated by up-regulation of the JNK pathway.

Description: Publication date: Available online 8 August 2015 Source: FEBS Open Bio Author(s): Jonghwan Lee, Do Won Hwang, Seung U Kim, Dong Soo Lee, Yong Seung Lee, Hyejung Heo, Bahy A. Ali, Abdulaziz A. Al-Khedhairy, Soonhag Kim Evaluation of the function of microRNAs (miRNAs or miRs) through miRNA expression profiles during neuronal differentiation plays a critical role not only in identifying unique miRNAs relevant to cellular development but also in understanding regulatory functions of the cell-specific miRNAs in living organisms. Here, we examined the microarray-based miRNA expression profiles of G2 cells (recently developed human neural stem cells) and monitored the expression pattern of known neuron-specific miR-9 and miR-124a during neuronal differentiation of G2 cells in vitro and in vivo . Of 500 miRNAs analyzed by microarray of G2 cells, the expression of 90 miRNAs was significantly increased during doxycycline-dependent neuronal differentiation of G2 cells and about 60 miRNAs showed a gradual enhancement of gene expression as neuronal differentiation progressed. Real-time PCR showed that expression of endogenous mature miR-9 was continuously and gradually increased in a pattern dependent on the period of neuronal differentiation of G2 cells while the increased expression of neuron-specific mature miR-124a was barely observed during neurogenesis. Our recently developed miRNA reporter imaging vectors (CMV/Gluc/3×PT_miR-9 and CMV/Gluc/3×PT_miR-124a) containing Gaussia luciferase, CMV promoter and three copies of complementary nucleotides of each corresponding miRNA showed that luciferase activity from CMV/Gluc/3×PT_miR-9 was gradually decreased both in vitro and in vivo in G2 cells induced to differentiate into neurons. However, in vitro and in vivo bioluminescence signals for CMV/Gluc/3×PT_miR-124a were not significantly different between undifferentiated and differentiated G2 cells. Our results demonstrate that biogenesis of neuron-specific miR-124a is not necessary for doxycycline-dependent neurogenesis of G2 cells.

Description: Publication date: Available online 7 August 2015 Source: FEBS Open Bio Author(s): Tao Liu, Xuan-Cheng Fang, Zhen Ding, Ze-Gan Sun, Li-Ming Sun, Yi-Lian Wang Inflammatory markers have been proposed to predict clinical outcomes in many types of cancers. The purpose of this study was to explore the influence of the lymphocyte-to-monocyte ratio (LMR) on clinical prognosis of patients with osteosarcoma. This study collected 327 patients who underwent surgical treatment for osteosarcoma during the period 2006 2010. LMR was calculated from pre-operative peripheral blood cells counts. The optimal cut-off value of LMR was determined based on receiver operating characteristic curve analysis. Overall survival (OS) and event free survival (EFS) was plotted using the Kaplan–Meier method and evaluated by the log-rank test. A predictive model was established to predict clinical prognosis for OS, and the predictive accuracy of this model was determined by concordance index (c-index). Our results showed that young age, elevated alkaline phosphatase, metastasis at diagnosis, chemotherapy, lymphocyte and monocyte counts were significantly associated with LMR. Low LMR was associated with shorter OS and EFS ( P 〈 0.001), and was an independent predictor of both OS and EFS (HR=1.72, 95% CI=1.14-2.60, P = 0.010; HR=1.89, 95% CI=1.32-2.57, P = 0.009). The nomogram performed well in the prediction of overall survival in patients with osteosarcoma (c-index 0.630). In conclusion, low pre-operative LMR is associated with a poor prognosis in patients suffering from osteosarcoma. A prospective study is warranted for further validation of our results.

Description: Publication date: Available online 11 August 2015 Source: FEBS Open Bio Author(s): Charlotte Harrison, K. Ravi Acharya Angiotensin converting enzyme (ACE) is a zinc-dependent dipeptidyl carboxypeptidase with an essential role in blood pressure homeostasis in mammals. ACE has long been targeted in the treatment of hypertension through ACE inhibitors, however current inhibitors are known to cause severe side effects. Therefore, there is a requirement for a new generation of ACE inhibitors and structural information will be invaluable in their development. ACE is a challenging enzyme to work with due to its extensive glycosylation. As such, the Drosophila melanogaster ACE homologue, AnCE, which shares ∼ 60% sequence similarity with human ACE, can be used as a model for studying inhibitor binding. The presence of ligands originating from the crystallisation condition at the AnCE active site has proved an obstacle to studying the binding of new inhibitor precursors. Here we present the crystal structure of AnCE (in a new crystal form) at 1.85 Å resolution, using crystals grown under different conditions. This new structure may be more suitable for studying the binding of new compounds, with the potential of developing a new generation of improved ACE inhibitors. Graphical abstract

Description: Nucleobase modifications dramatically alter nucleic acid structure and thermodynamics. 2-thiouridine (s 2 U) is a modified nucleobase found in tRNAs and known to stabilize U:A base pairs and destabilize U:G wobble pairs. The recently reported crystal structures of s 2 U-containing RNA duplexes do not entirely explain the mechanisms responsible for the stabilizing effect of s 2 U or whether this effect is entropic or enthalpic in origin. We present here thermodynamic evaluations of duplex formation using ITC and UV thermal denaturation with RNA duplexes containing internal s 2 U:A and s 2 U:U pairs and their native counterparts. These results indicate that s 2 U stabilizes both duplexes. The stabilizing effect is entropic in origin and likely results from the s 2 U-induced preorganization of the single-stranded RNA prior to hybridization. The same preorganizing effect is likely responsible for structurally resolving the s 2 U:U pair-containing duplex into a single conformation with a well-defined H-bond geometry. We also evaluate the effect of s 2 U on single strand conformation using UV- and CD-monitored thermal denaturation and on nucleoside conformation using 1 H NMR spectroscopy, MD and umbrella sampling. These results provide insights into the effects that nucleobase modification has on RNA structure and thermodynamics and inform efforts toward improving both ribozyme-catalyzed and nonenzymatic RNA copying.