Norshariza Nordin

Receptor for Advanced Glycation End Products and Its Involvement in Inflammatory Diseases

The receptor for advanced glycation end products (RAGE) is a transmembrane receptor of the immunoglobulin superfamily, capable of binding a broad repertoire of ligands. RAGE-ligands interaction induces a series of signal transduction cascades and lead to the activation of transcription factor NF-κB as well as increased expression of cytokines, chemokines, and adhesion molecules. These effects endow RAGE with the role in the signal transduction from pathogen substrates to cell activation during the onset and perpetuation of inflammation. RAGE signaling and downstream pathways have been implicated in a wide spectrum of inflammatory-related pathologic conditions such as arteriosclerosis, Alzheimers disease, arthritis, acute respiratory failure, and sepsis. Despite the significant progress in other RAGE studies, the functional importance of the receptor in clinical situations and inflammatory diseases still remains to be fully realized. In this review, we will summarize current understandings and lines of evidence on the molecular mechanisms through which RAGE signaling contributes to the pathogenesis of the aforementioned inflammation-associated conditions.

Advancements in reprogramming strategies for the generation of induced pluripotent stem cells

Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells has emerged as an invaluable method for generating patient-specific stem cells of any lineage without the use of embryonic materials. Following the first reported generation of iPS cells from murine fibroblasts using retroviral transduction of a defined set of transcription factors, various new strategies have been developed to improve and refine the reprogramming technology. Recent developments provide optimism that the generation of safe iPS cells without any genomic modification could be derived in the near future for the use in clinical settings. This review summarizes current and evolving strategies in the generation of iPS cells, including types of somatic cells for reprogramming, variations of reprogramming genes, reprogramming methods, and how the advancement iPS cells technology can lead to the future success of reproductive medicine.

A bismuth diethyldithiocarbamate compound promotes apoptosis in HepG2 carcinoma, cell cycle arrest and inhibits cell invasion through modulation of the NF-κB activation pathway

The compound with R=CH2CH3 in Bi(S2CNR2)3 (1) is highly cytotoxic against a range of human carcinoma, whereas that with R=CH2CH2OH (2) is considerably less so. Both 1 and 2 induce apoptosis in HepG2 cells with some evidence for necrosis induced by 2. Based on DNA fragmentation, caspase activities and human apoptosis PCR-array analysis, both the extrinsic and intrinsic pathways of apoptosis have been shown to occur. While both compounds activate mitochondrial and FAS apoptotic pathways, compound 1 was also found to induce another death receptor-dependent pathway by induction of CD40, CD40L and TNF-R1 (p55). Further, 1 highly expressed DAPK1, a tumour suppressor, with concomitant down-regulation of XIAP and NF-κB. Cell cycle arrest at the S and G2/M phases correlates with the inhibition of the growth of HepG2 cells. The cell invasion rate of 2 is 10-fold higher than that of 1, a finding correlated with the down-regulation of survivin and XIAP expression by 1. Compounds 1 and 2 interact with DNA through different binding motifs with 1 interacting with AT- or TA-specific sites followed by inhibition of restriction enzyme digestion; 2 did not interfere with any of the studied restriction enzymes.

Expression Profiles of Wnt Genes during Neural Differentiation of Mouse Embryonic Stem Cells

The Wnt family of secreted signaling proteins regulates many aspects of animal development and the behavior of several types of stem cells, including embryonic stem (ES) cells. Activation of canonical Wnt signaling has been shown to either inhibit or promote the differentiation of ES cells into neurons, depending on the stage of differentiation. Here, we describe the expression of all 19 mouse Wnt genes during this process. Using the well-established retinoic acid induction protocol we found that all Wnt genes except Wnt8b are expressed as ES cells differentiate into neurons, many of them in dynamic patterns. The expression pattern of 12 Wnt genes was analyzed quantitatively at 2-day intervals throughout neural differentiation, showing that multiple Wnt genes are expressed at each stage. A large proportion of these, including both canonical and noncanonical Wnts, are expressed at highest levels during later stages of differentiation. The complexity of the patterns observed indicates that disentangling specific roles for individual Wnt genes in the differentiation process will be a significant challenge.

A plethora of human pluripotent stem cells

At the early stages of mammalian development, a number of developmentally plastic cells appear that possess the ability to give rise to all of the differentiated cell types normally derived from the three primary germ layers – unique character known as pluripotency. To date, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been shown to be truly pluripotent. However, recent studies have revealed a variety of other cells that demonstrate pluripotentiality, including very small embryonic‐like stem cells (VSELs), amniotic fluid stem cells (AFSCs), marrow‐isolated adult multilineage inducible cells (MIAMI) and multipotent adult precursor cells (MAPCs). This review summarises key features of these six kinds of pluripotent and potentially pluripotent stem cells (ESCs, iPSCs, VSELs, AFSCs, MIAMI and MAPCs) and the evidence for their pluripotency properties.

Mesenchymal stem cells: From stem cells to sarcomas.

Mesenchymal stem cells (MSCs) have garnered vast interests in clinical settings, especially in regenerative medicine due to their unique properties—they are reliably isolated and expanded from various tissue sources; they are able to differentiate into mesodermal tissues such as bones, cartilages, adipose tissues, and muscles; and they have unique immunosuppressive properties. However, there are some concerns pertaining to the role of MSCs in the human body. On one hand, they are crucial component in the regeneration and repair of the human body. On the contrary, they are shown to transform into sarcomas. Although the exact mechanisms are still unknown, many new leads have pointed to the belief that MSCs do play a role in sarcomagenesis. This review focuses on the current updates and findings of the role of MSCs in their transformation process into sarcomas.

Epigenetic modification does not determine the time of POU5F1 transcription activation in cloned bovine embryos

PurposeTo investigate the effect of epigenetic modification on pattern, time and capacity of transcription activation of POU5F1, the key marker of pluripotency, in cloned bovine embryos.MethodsBovine fibroblasts were stably transfected with POU5F1 promoter-driven enhanced green fluorescent protein (EGFP). This provided a visible marker to investigate the effect of post-activation treatment of cloned bovine embryos with trichostatin A (TSA) on time and capacity of POU5F1 expression and its subsequent effect on in vitro development of cloned bovine embryos.ResultsIrrespective of TSA treatment, POU5F1 expression was not detected until 8–16 cell stage, but was detected in both inner cell mass and trophectoderm at the blastocyst stage. TSA treatment significantly increased POU5F1 expression, and the yield and quality of cloned embryo development compared to control.ConclusionThe POU5F1 expression of cloned embryos is strictly controlled by the stage of embryo development and may not be altered by TSA-mediated changes occur in DNA-methylation and histone-acetylation of the genome.

Lentivirus vector driven by polybiquitin C promoter without woodchuck posttranscriptional regulatory element and central polypurine tract generates low level and short-lived reporter gene expression

Lentivirus (LV) encoding woodchuck posttranscriptional regulatory element (WPRE) and central polypurine tract (cPPT) driven by CMV promoter have been proven to act synergistically to increase both transduction efficiency and gene expression. However, the inclusion of WPRE and cPPT in a lentiviral construct may pose safety risks when administered to human. A simple lentiviral construct driven by an alternative promoter with proven extended duration of gene expression without the two regulatory elements would be free from the risks. In a non-viral gene delivery context, gene expression driven by human polybiquitin C (UbC) promoter resulted in higher and more persistent expression in mouse as compared to cytomegalovirus (CMV) promoter. In this study, we measured the efficiency and persistency of green fluorescent protein (GFP) reporter gene expression in cells transduced with LV driven by UbC (LV/UbC/GFP) devoid of the WPRE and cPPT in comparison to the established LV construct encoding WPRE and cPPT, driven by CMV promoter (LV/CMV/GFP). However, we found that LV/UbC/GFP was inferior to LV/CMV/GFP in many aspects: (i) the titer of virus produced; (ii) the levels of reporter gene expression when MOI value was standardized; and (iii) the transduction efficiency in different cell types. The duration of reporter gene expression in selected cell lines was also determined. While the GFP expression in cells transduced with LV/CMV/GFP persisted throughout the experimental period of 14 days, expression in cells transduced with LV/UbC/GFP declined by day 2 post-transduction. In summary, the LV driven by the UbC promoter without the WPRE and cPPT does not exhibit enhanced or durable transgene expression.

Isothiocyanate from Moringa oleifera seeds mitigates hydrogen peroxide-induced cytotoxicity and preserved morphological features of human neuronal cells

Reactive oxygen species are well known for induction of oxidative stress conditions through oxidation of vital biomarkers leading to cellular death via apoptosis and other process, thereby causing devastative effects on the host organs. This effect is believed to be linked with pathological alterations seen in several neurodegenerative disease conditions. Many phytochemical compounds proved to have robust antioxidant activities that deterred cells against cytotoxic stress environment, thus protect apoptotic cell death. In view of that we studied the potential of glucomoringin-isothiocyanate (GMG-ITC) or moringin to mitigate the process that lead to neurodegeneration in various ways. Neuroprotective effect of GMG-ITC was performed on retinoic acid (RA) induced differentiated neuroblastoma cells (SHSY5Y) via cell viability assay, flow cytometry analysis and fluorescence microscopy by means of acridine orange and propidium iodide double staining, to evaluate the anti-apoptotic activity and morphology conservation ability of the compound. Additionally, neurite surface integrity and ultrastructural analysis were carried out by means of scanning and transmission electron microscopy to assess the orientation of surface and internal features of the treated neuronal cells. GMG-ITC pre-treated neuron cells showed significant resistance to H2O2-induced apoptotic cell death, revealing high level of protection by the compound. Increase of intracellular oxidative stress induced by H2O2 was mitigated by GMG-ITC. Thus, pre-treatment with the compound conferred significant protection to cytoskeleton and cytoplasmic inclusion coupled with conservation of surface morphological features and general integrity of neuronal cells. Therefore, the collective findings in the presence study indicated the potentials of GMG-ITC to protect the integrity of neuron cells against induced oxidative-stress related cytotoxic processes, the hallmark of neurodegenerative diseases.

In Silico Prediction and Validation of Gfap as an miR-3099 Target in Mouse Brain

Abstract MicroRNAs are small non-coding RNAs that play crucial roles in the regulation of gene expression and protein synthesis during brain development. MiR-3099 is highly expressed throughout embryogenesis, especially in the developing central nervous system. Moreover, miR-3099 is also expressed at a higher level in differentiating neurons in vitro, suggesting that it is a potential regulator during neuronal cell development. This study aimed to predict the target genes of miR-3099 viain-silico analysis using four independent prediction algorithms (miRDB, miRanda, TargetScan, and DIANA-micro-T-CDS) with emphasis on target genes related to brain development and function. Based on the analysis, a total of 3,174 miR-3099 target genes were predicted. Those predicted by at least three algorithms (324 genes) were subjected to DAVID bioinformatics analysis to understand their overall functional themes and representation. The analysis revealed that nearly 70% of the target genes were expressed in the nervous system and a significant proportion were associated with transcriptional regulation and protein ubiquitination mechanisms. Comparison of in situ hybridization (ISH) expression patterns of miR-3099 in both published and in-house-generated ISH sections with the ISH sections of target genes from the Allen Brain Atlas identified 7 target genes (Dnmt3a, Gabpa, Gfap, Itga4, Lxn, Smad7, and Tbx18) having expression patterns complementary to miR-3099 in the developing and adult mouse brain samples. Of these, we validated Gfap as a direct downstream target of miR-3099 using the luciferase reporter gene system. In conclusion, we report the successful prediction and validation of Gfap as an miR-3099 target gene using a combination of bioinformatics resources with enrichment of annotations based on functional ontologies and a spatio-temporal expression dataset.