Expression of microRNA-1, microRNA-133a and Hand2 protein in cultured embryonic rat cardiomyocytes

In this study, we investigated the expression of the pathway, SRF–microRNA-1/microRNA-133a–Hand2, in the Wistar rat embryonic ventricular cardiomyocytes under conventional monolayer culture. The morphological observation of the cultured cardiomyocytes and the mRNA expression levels of three vital constituent proteins, MLC-2v, N-cadherin, and connexin43, demonstrated the immaturity of these cultured cells, which was featured by less myofibril density, immature sarcomeric structure, and significantly lower mRNA expression of the three constituent proteins than those in neonatal ventricular samples. More importantly, results in this study suggest that the change of SRF–microRNA-1/microRNA-133a–Hand2 pathway results into the attenuation of the Hand2 repression in cultured cardiomyocytes. These outcomes are valuable to understand the cellular state as embryonic cardiomyocytes to be in vitro model and might be useful for the assessment of engineered cardiac tissue and cardiac differentiation of stem cells.     

Identification of microRNAs specific for high producer CHO cell lines using steady-state cultivation

MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene expression. Hence, microRNAs are considered as potential targets for engineering of Chinese hamster ovary (CHO) cells to improve recombinant protein production. Here, we analyzed and compared the microRNA expression patterns of high, low, and non-producing recombinant CHO cell lines expressing two structurally different model proteins in order to identify microRNAs that are involved in heterologous protein synthesis and secretion and thus might be promising targets for cell engineering to increase productivity. To generate reproducible and comparable data, the cells were cultivated in a bioreactor under steady-state conditions. Global microRNA expression analysis showed that mature microRNAs were predominantly upregulated in the producing cell lines compared to the non-producer. Several microRNAs were significantly differentially expressed between high and low producers, but none of them commonly for both model proteins. The identification of target messenger RNAs (mRNAs) is essential to understand the biological function of microRNAs. Therefore, we negatively correlated microRNA and global mRNA expression data and combined them with computationally predicted and experimentally validated targets. However, statistical analysis of the identified microRNA-mRNA interactions indicated a considerable false positive rate. Our results and the comparison to published data suggest that the reaction of CHO cells to the heterologous protein expression is strongly product- and/or clone-specific. In addition, this study highlights the urgent need for reliable CHO-specific microRNA target prediction tools and experimentally validated target databases in order to facilitate functional analysis of high-throughput microRNA expression data in CHO cells.     

Circulating MicroRNA Biomarkers for Glioma and Predicting Response to Therapy

The need for glioma biomarkers with improved sensitivity and specificity has sparked research into short non-coding RNA known as microRNA (miRNA). Altered miRNA biogenesis and expression in glioma plays a vital role in important signaling pathways associated with a range of tumor characteristics including gliomagenesis, invasion, and malignancy. This review will discuss current research into the role of miRNA in glioma and altered miRNA expression in biofluids as candidate biomarkers with a particular focus on glioblastoma, the most malignant form of glioma. The isolation and characterization of miRNA using cellular and molecular biology techniques from the circulation of glioma patients could potentially be used for improved diagnosis, prognosis, and treatment decisions. We aim to highlight the links between research into miRNA function, their use as biomarkers, and how these biomarkers can be used to predict response to therapy. Furthermore, increased understanding of miRNA in glioma biology through biomarker research has led to the development of miRNA therapeutics which could restore normal miRNA expression and function and improve the prognosis of glioma patients. A panel of important miRNA biomarkers for glioma in various biofluids discovered to date has been summarized here. There is still a need, however, to standardize techniques for biomarker characterization to bring us closer to clinically relevant miRNA-based diagnostic and therapeutic signatures. A clinically validated biomarker panel has potential to improve time to diagnosis, predicting response to treatment and ultimately the prognosis of glioma patients.     

Quercetin Potentiates the Antitumor Activity of Rituximab in Diffuse Large B-Cell Lymphoma by Inhibiting STAT3 Pathway

STAT3 pathway plays an important role in the growth of diffuse large B-cell lymphoma (DLBCL) cells. Here we investigated the antitumor activity of Quercetin, a flavonoid compound, in combination with rituximab in DLBCL cell lines in vitro. We found that Quercetin synergistically enhanced rituximab-induced growth inhibition and apoptosis in DLBCL cell lines. Moreover, we found Quercetin exerted inhibitory activity against STAT3 pathway and downregulated the expression of survival genes. These results suggest that combining the Quercetin with rituximab may present an attractive and potentially effective way for the treatment of DLBCL.     

Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis

The tumor suppressor PTEN is a lipid phosphatase that is found mutated in different types of human cancers. PTEN suppresses cell proliferation by inhibiting the PI3K-Akt signaling pathway at the cell membrane. However, PTEN is also demonstrated to localize in the cell nucleus where it exhibits tumor suppressive activity via a different, unknown mechanism. In this study we report that PTEN also localizes to the nucleolus and that nucleolar PTEN plays an important role in regulating nucleolar homeostasis and maintaining nucleolar morphology. Overexpression of nuclear PTEN in PTEN null cells inhibits Akt phosphorylation and reduces cell size. Knockdown of PTEN in PTEN positive cells leads to nucleolar morphologic changes and an increase in the proportion of cells with a greater number of nucleoli. In addition, knockdown of PTEN in PTEN positive cells increased ribosome biogenesis. These findings expand current understanding of function and relevance of nuclear localized PTEN and provide a foundation for the development of novel therapies targeting PTEN.     

MicroRNA-2 Suppresses Lewis Lung Cancer Cells Proliferation,Invasion, and Migration in Tumor-Bearing Mice

We sought to find the biological effects of MicroRNA-2 in suppressing Lewis lung cancer cells proliferation, invasion, and migration in tumor-bearing mice. MicroRNA-2 was transfected into Lewis lung cancer cells of tumor-bearing mice by gene transient transfection technique and these Lewis-microRNA-2 cells were taken as MicroRNA transfection group. At the same time, Lewis cells were taken as control group and Lewis-EGFP cells as empty plasmid group. The growth curves of cells in the three groups were drawn by manual counting method, while the invasiveness of cells in the three groups was compared by transmembrane cell invasion assay. The three kinds of cells were seeded into BALB/Nude SPF level nude mice to detect the formation of tumors and the number of metastases by Xenograft experiments. The result showed that the MicroRNA transfection group has the lowest vitality of cells proliferation, fewest cells passed through matrigel matrix protein layer, and lowest cells invasive rate. Mice with Lewis-microRNA-2 cells apparently had a longer time of tumor formation. The average tumor mass and the number of metastases were significantly lower than the other two groups. MicroRNA-2 significantly inhibited Lewis lung cancer cell proliferation, invasion and migration in tumor-bearing mice, which may be associated with the regulation of target genes PLK1 and TGF-β.     

Primary alveolar macrophages exposed to diesel particulate matter increase RAGE expression and activate RAGE signaling

Receptors for advanced glycation end-products (RAGE) are members of the immunoglobulin superfamily of cell-surface receptors implicated in mechanisms of pulmonary inflammation. In the current study, we test the hypothesis that RAGE mediates inflammation in primary alveolar macrophages (AMs) exposed to diesel particulate matter (DPM). Quantitative RT-PCR and immunoblotting revealed that RAGE was up-regulated in Raw264.7 cells, an immortalized murine macrophage cell line and primary AMs exposed to DPM for 2 h. Because DPM increased RAGE expression, we exposed Raw264.7 cells and primary AMs isolated from RAGE null and wild-type (WT) mice to DPM prior to the assessment of inflammatory signaling intermediates. DPM led to the activation of Rat sarcoma GTPase (Ras), p38 MAPK and NF-κB in WT AMs and, when compared to WT AMs, these intermediates were diminished in DPM-exposed AMs isolated from RAGE null mice. Furthermore, cytokines implicated in inflammation, including IL-4, IL-12, IL-13 and TNFα, were all significantly decreased in DPM-exposed RAGE null AMs compared to similarly exposed WT AMs. These results demonstrate that diesel-induced inflammatory responses by primary AMs are mediated, at least in part, via RAGE signaling mechanisms. Further work may show that RAGE signaling in both alveolar epithelial cells and resident macrophages is a potential target in the treatment of inflammatory lung diseases exacerbated by environmental pollution.     

Preparation of cinnamoyl-CoA and analysis of the enzyme activity of recombinant CCR protein from Populus tomentosa

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44), which catalyzes the reduction of cinnamoyl-CoA esters to their respective cinnamaldehydes, is considered as a key enzyme in lignin formation. The substrates of CCR, cinnamoyl-CoA esters, are products of 4-Coumarate-CoA ligase (4CL, EC 6.2.1.12), which is an enzyme upstream of CCR. The PtCCR and Pt4CL were isolated from Populus tomentosa and expressed in E. coli. Results showed that 4CL can catalyze the conversion of hydroxycinnamic acids to cinnamoyl-CoA esters, with high efficiency. The purification of esters using SPE cartridges suggested that 40 % methanol with 0.1 M of acetic acid was the optimal elution buffer for cinnamoyl-CoA esters. The optimization of prokaryotic expression demonstrated that the best expression conditions for recombinant PtCCR was 6 h of 0.4 mM IPTG induction at 37 °C. PtCCR enzyme assay illustrated that the recombinant protein can catalyze the reduction of cinnamoyl-CoA esters. Kinetics analysis showed that feruloyl-CoA has higher affinity to PtCCR with faster reaction speed (Vmax), indicating that feruloyl-CoA was the most favorable substrate for PtCCR catalysis. The recombinant protein was expressed in E. coli, purified through affinity column chromatography, and characterized by SDS-PAGE. SPE cartridges were used to purify the ester products of the Pt4CL reaction. HPLC-MS was used to analyze the structure of esters and evaluate their purity or quantity. Furthermore, the enzyme activity of recombinant CCR to feruloyl-CoA at different pHs indicated that compartmentalization may be an important factor in lignin monomer formation.     

Relative mRNA expression and immunolocalization for transforming growth factor-beta (TGF-β) and their effect on in vitro development of caprine preantral follicles

This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-β) and its receptors (TGF-βRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-β, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-β and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-β receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100 ng/ml), TGF-β (10 ng/ml), or TGF-β + FSH for 18 d. TGF-β increased significantly oocyte diameter when compared to FSH alone and control. After 18 d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-β in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-β and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.     

Effect of Recombinant hPTEN Gene Expression on PDGF Induced VSMC Proliferation

Human phosphatase and tensin homolog (hPTEN) gene was expressed in vascular smooth muscle cells (VSMCs) to study its effect on VSMC proliferation induced in platelet-derived growth factor (PDGF) conditioned medium. After G418 selection, MTT assay was conducted to examine transfected VSMC proliferation induced in human PDGF conditioned medium. We successfully constructed eukaryotic expression vector pcDNA4/myc-His-PTEN and transferred into VSMC cells. We report that in vitro proliferation of VSMC was inhibited in PTEN transfected VSMCs induced in PDGF conditioned medium. RT-PCR and Western blot results indicated significantly high levels of protein kinase B-PKB and nuclear factor kappa B mRNA and protein, respectively, in PDGF group as compared with the control group.     

Implementation of Transportation Distance for Analyzing FLIM and FRET Experiments

Analysis of fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET) experiments in living cells is usually based on mean lifetimes computations. However, these mean lifetimes can induce misinterpretations. We propose in this work the implementation of the transportation distance for FLIM and FRET experiments in vivo. This non-fitting indicator, which is easy to compute, reflects the similarity between two distributions and can be used for pixels clustering to improve the estimation of the FRET parameters. We study the robustness and the discriminating power of this transportation distance, both theoretically and numerically. In addition, a comparison study with the largely used mean lifetime differences is performed. We finally demonstrate practically the benefits of the transportation distance over the usual mean lifetime differences for both FLIM and FRET experiments in living cells.     

GABA Pharmacology: The Search for Analgesics

Decades of research have been devoted to defining the role of GABAergic transmission in nociceptive processing. Much of this work was performed using rigid, orthosteric GABA analogs created by Povl Krogsgaard-Larsen and his associates. A relationship between GABA and pain is suggested by the anatomical distribution of GABA receptors and the ability of some GABA agonists to alter nociceptive responsiveness. Outlined in this report are data supporting this proposition, with particular emphasis on the anatomical localization and function of GABA-containing neurons and the molecular and pharmacological properties of GABA_A and GABA_B receptor subtypes. Reference is made to changes in overall GABAergic tone, GABA receptor expression and activity as a function of the duration and intensity of a painful stimulus or exposure to GABAergic agents. Evidence is presented that the plasticity of this receptor system may be responsible for the variability in the antinociceptive effectiveness of compounds that influence GABA transmission. These findings demonstrate that at least some types of persistent pain are associated with a regionally selective decline in GABAergic tone, highlighting the need for agents that enhance GABA activity in the affected regions without compromising GABA function over the long-term. As subtype selective positive allosteric modulators may accomplish these goals, such compounds might represent a new class of analgesic drugs.     

Suppression of PI3K/Akt signaling by synthetic bichalcone analog TSWU-CD4 induces ER stress- and Bax/Bak-mediated apoptosis of cancer cells

Suppression of the activity of pro-apoptotic Bcl-2-family proteins frequently confers chemoresistance to many human cancer cells. Using subcellular fractionation, the ER calcium (Ca⁺⁺) channel inhibitor dantrolene and small interfering RNA (siRNA) against Bax or Bak, we show that the new synthetic bichalcone analog TSWU-CD4 induces apoptosis in human cancer cells by releasing endoplasmic reticulum (ER)-stored Ca⁺⁺ through ER/mitochondrial oligomerization of Bax/Bak. Blockade of the protein kinase RNA-like ER kinase or the unfolded protein response regulator glucose-regulated protein 78 expression by siRNA not only suppressed oligomeric Bax/Bak-mediated pro-caspase-12 cleavage and apoptosis but also resulted in an inhibition of Bcl-2 downregulation induced by TSWU-CD4. Induction of the ER oligomerization of Bax/Bak and apoptosis by TSWU-CD4 were suppressed by Bcl-2 overexpression. Inhibition of lipid raft-associated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling by TSWU-CD4 induced ER stress- and oligomeric Bax/Bak-mediated apoptosis, which were substantially reversed by overexpression of the wt PI3K p85α subunit. Taken together, these results suggest that suppression of lipid raft-associated PI3K/Akt signaling is required for the ER stress-mediated apoptotic activity of Bax/Bak, which is responsible for the ability of TSWU-CD4-treated cancer cells to exit the ER-mitochondrial apoptotic cell death pathway.     

Progression of O6-methylguanine-DNA methyltransferase and temozolomide resistance in cancer research

Temozolomide (TMZ) is an alkylating agent that is widely used in chemotherapy for cancer. A key mechanism of resistance to TMZ is the overexpression of O⁶-methylguanine-DNA methyltransferase (MGMT). MGMT specifically repairs the DNA O⁶-methylation damage induced by TMZ and irreversibly inactivates TMZ. Regulation of MGMT expression and research regarding the mechanism of TMZ resistance will help rationalize the clinical use of TMZ. In this review, we provide an overview of recent advances in the field, with particular emphasis on MGMT structure, function, expression regulation, and the association between MGMT and resistance to TMZ.     

Optimization of conditions for expression of recombinant interferon-γ in E.coli

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.     

Calcitonin Gene-Related Peptide Inhibits Osteolytic Factors Induced by Osteoblast In Co-Culture System with Breast Cancer

Recently, it was found that α-Calcitonin gene-related peptide (CGRP) was associated with breast cancer metastases, but the role of CGRP in interaction between breast cancer and osteoblast during bone metastases is not clear. Here, we investigated the effect of CGRP on osteoblast in co-culture system with breast cancer. Using a breast cancer–osteoblast co-culture system, we chose MDA-MB-231 for breast cancer and human cell line MG-63 for osteoblast. CGRP was added to this co-culture system. The expression levels of the Runx2, RANK1, and osteoprotegerin (OPG) were analyzed using real-time PCR and western blot. CGRP receptors were investigated by immunofluorescence. We found that breast cancer cells cause osteolysis lesions by upregulating Runx2 expression, decreasing OPG expression, and increasing RANKL expression in osteoblasts. Our data prove that CGRP can regulate osteoclast coupling genes in osteoblast by increasing OPG, and decreasing RANKL and Runx2 expressions in a time-dependent manner; and inhibit those osteolytic factors induced by interaction between breast cancer cells and osteoblast. This inhibition could be abolished by the CGRP antagonist, CGRP8–37. In conclusion, calcitonin receptor-like receptor is the key player for CGRP’s effect in this co-culture system.     

Transarterial chemoembolization combined with sorafenib for unresectable hepatocellular carcinoma: a systematic review and meta-analysis

Sorafenib in combination with Transarterial chemoembolization (TACE) is increasingly used in patients with unresectable hepatocellular carcinoma (HCC), but the current evidence is still controversial. The aim of this systematic review was to evaluate the effectiveness and safety of TACE plus sorafenib versus TACE alone for unresectable HCC. We searched PubMed, EMBASE and the Cochrane Library for clinical trials comparing TACE plus sorafenib with TACE alone for unresectable HCC. The study outcomes included overall survival (OS), time to progression (TTP), objective response and adverse events (AEs). Six studies including 1,181 patients were included. Meta-analysis of all studies suggested that the combination therapy group had significant longer OS than TACE group [hazard ratio (HR) = 0.64, 95 % confidence interval (CI) = 0.43–0.97], but the pooled HR of randomized controlled trials (RCTs) failed to achieve statistical significance. For TTP, meta-analysis in both RCTs subgroup and retrospective studies subgroup suggested that combination therapy was superior to TACE group. The combination therapy was also associated with better response to treatment (risk ratio = 1.45, 95 % CI = 1.04–2.02) when both RCTs and retrospective studies were pooled. However, the sorafenib associated AEs were more frequent in the combination therapy group. In conclusion, the combination of TACE and sorafenib is likely to improve OS, TTP and response to treatment when compared with TACE monotherapy. The combination group is also associated with more sorafenib-related AEs.     

Intromitochondrial IκB/NF-κB signaling pathway is involved in amyloid β peptide-induced mitochondrial dysfunction

Mitochondrial dysfunction is a hallmark of amyloid β peptide (Aβ)-induced neuronal toxicity in Alzheimer’s disease (AD). However, the underlying mechanism (s) of Aβ-induced mitochondrial dysfunction is still not fully understood. There is evidence that nuclear factor-κB (NF-κB) is involved in Aβ-induced neurotoxicity and is present in mitochondria. Using HT22 murine hippocampal neuronal cells and isolated mitochondria, the present study investigated whether intramitochondrial inhibitor of NF-κB (IκB)/NF-κB signaling pathway was involved in mitochondrial dysfunction induced by Aβ. It was found that Aβ impaired mitochondrial function through a NF-κB-dependent signaling pathway. Intramitochondrial IκBα/NF-κB pathway, induced by Aβ, decreased the expression of cytochrome c oxidase subunit (COXIII) and inhibited COX activity. These results provide new insights into the mechanism underlying the neurotoxic effect of Aβ and open up new therapeutic perspectives for AD.