Abstract: Sphingomyelin is found in the cell membrane of all eukaryotic cells, and was for a long time considered merely as a structural component. However, during the last two decades, metabolites of sphingomyelin, especially sphingosine 1-phosphate (S1P), have proven to be physiologically significant regulators of cell function. Through its five different G protein-coupled receptors, S1P regulates a wide array of cellular processes, ranging from stimulating cellular proliferation and migration, to the inhibition of apoptosis and induction of angiogenesis and modulation of cellular calcium homeostasis. Many of the processes regulated by S1P are important for normal cell physiology, but may also induce severe pathological conditions, especially in malignancies like cancer. Thus, understanding S1P signaling mechanisms has been the aim of a multitude of investigations. Great interest has also been shown in understanding the action of sphingosine kinase (SphK), i.e., the kinase phosphorylating sphingosine to S1P, and the interactions between S1P and growth factor signaling. In the present review, we will discuss recent findings regarding the possible importance of S1P and SphK in the etiology of thyroid cancer. Although clinical data is still scarce, our in vitro findings suggest that S1P may function as a “double-edged sword”, as the receptor profile of thyroid cancer cells largely determines whether S1P stimulates or blocks cellular migration. We will also discuss the interactions between S1P- and VEGF-evoked signaling, and the importance of a S1P1-VEGF receptor 2 complex in thyroid cancer cells.
Abstract: Since the discovery of microRNA (miRNA), the polymorphisms that affect miRNA regulation had been extensively investigated by many independent studies. Recently, researchers utilized bioinformatics and statistical approaches for genome-wide analysis on the human polymorphisms that reside in the miRNA genes, targets, and/or genes involved in miRNA processing. In this review, we will give an overview about the important findings of these studies from three perspectives: architecture of the polymorphisms within miRNAs or their targets, potential functional consequences of the polymorphisms on miRNA processing or targeting, and the associations of the polymorphisms with miRNA or target gene expression. The results of the previous studies demonstrated the signatures of natural selections on the miRNA genes and their targets, and proposed a collection of potentially functional, expression-associated, and/or positively selected polymorphisms that are promising for further investigations. In the meantime, a few useful resources about the polymorphic miRNA regulation have been developed and the different features of these databases were discussed in this review. Though recent research had benefited from these comprehensive studies and resources, there were still gaps in our knowledge about the polymorphisms involved in miRNA regulation, and future investigations were expected to address these questions.
Abstract: Glycoproteomics has emerged as a prime area of interest within the field of proteomics because glycoproteins have been shown to function as biomarkers for disease and as promising therapeutic targets. A significant challenge in the study of glycoproteins is the fact that they are expressed in relatively low abundance in cells. In response, various enrichment methods have been developed to improve the detection of glycoproteins. One such method involves their capture via oxidation of their glycan chains and covalent attachment with hydrazide resins which, when catalyzed by PNGase F, release N-linked glycans and convert the glycosite Asn to Asp; this conversion is identifiable with LC/ESI-MS/MS as a corresponding increase of 0.984 Da in molecular weight. The present study builds on this body of work, providing evidence of three additional strategies that improve glycoprotein identification: (1) use of a high resolution mass spectrometer—the Q Exactive MS—which delivers 2–3 times more glycoprotein identifications than a low resolution MS; (2) optimization of instrument settings and database search parameters to reduce misidentification of N-linked glycopeptides to ~1 percent; and (3) labeling glycopeptides with 18O during PNGase F treatment to locate N-linked glycosites within peptides containing multiple N-linked sequons.
Abstract: Poly (2-hydroxyethyl methacrylate) (HEMA) has been used as a clinical material, in the form of a soft hydrogel, for various surgical procedures, including endovascular surgery of liver. It is a clear liquid compound and, as a soft, flexible, water-absorbing material, has been used to make soft contact lenses from small, concave, spinning molds. Primary rat hepatocyte spheroids were created on a poly-HEMA-coated surface with the intention of inducing hepatic tissue formation and improving liver functions. We investigated spheroid formation of primary adult rat hepatocyte cells and characterized hepatic-specific functions under the special influence of fetal calf serum (FCS) and nonparencymal cells (NPC) up to six days in different culture systems (e.g., hepatocytes + FCS, hepatocytes – FCS, NPC + FCS, NPC – FCS, co-culture + FCS, co-culture – FCS) in both the spheroid model and sandwich model. Immunohistologically, we detected gap junctions, Ito cell/Kupffer cells, sinusoidal endothelial cells and an extracellular matrix in the spheroid model. FCS has no positive effect in the sandwich model, but has a negative effect in the spheroid model on albumin production, and no influence in urea production in either model. We found more cell viability in smaller diameter spheroids than larger ones by using the apoptosis test. Furthermore, there is no positive influence of the serum or NPC on spheroid formation, suggesting that it may only depend on the physical condition of the culture system. Since the sandwich culture has been considered a “gold standard” in vitro culture model, the hepatocyte spheroids generated on the poly-HEMA-coated surface were compared with those in the sandwich model. Major liver-specific functions, such as albumin secretion and urea synthesis, were evaluated in both the spheroid and sandwich model. The synthesis performance in the spheroid compared to the sandwich culture increases approximately by a factor of 1.5. Disintegration of plasma membranesin both models was measured by lactate dehydrogenase(LDH) release in both models. Additionally, diazepamwas used as a substrate in drug metabolism studies to characterize the differences in the biotransformation potential with metabolite profiles in both models. It showed that the diazepam metabolism activities in the spheroid model is about 10-fold lower than the sandwich model. The poly-HEMA-based hepatocyte spheroid is a promising new platform towards hepatic tissue engineering leading to in vitro hepatic tissue formation.
Abstract: The recent advent of high-throughput approaches has revealed widespread transcription of the human genome, leading to a new appreciation of transcription regulation, especially from noncoding regions. Distinct from most coding and small noncoding RNAs, long noncoding RNAs (lncRNAs) are generally expressed at low levels, are less conserved and lack protein-coding capacity. These intrinsic features of lncRNAs have not only hampered their full annotation in the past several years, but have also generated controversy concerning whether many or most of these lncRNAs are simply the result of transcriptional noise. Here, we assess these intrinsic features that have challenged lncRNA discovery and further summarize recent progress in lncRNA discovery with integrated methodologies, from which new lessons and insights can be derived to achieve better characterization of lncRNA expression regulation. Full annotation of lncRNA repertoires and the implications of such annotation will provide a fundamental basis for comprehensive understanding of pervasive functions of lncRNAs in biological regulation.
Abstract: A large variety of glycans is intricately located on the cell surface, and the overall profile (the glycome, given the entire repertoire of glycoconjugate-associated sugars in cells and tissues) is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that control cell-cell adhesion, immune response, microbial pathogenesis and other cellular events. The glycomic profile also reflects cellular alterations, such as development, differentiation and cancerous change. A glycoconjugate-based approach would therefore be expected to streamline discovery of novel cellular biomarkers. Development of such an approach has proven challenging, due to the technical difficulties associated with the analysis of various types of cellular glycomes; however, recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various classes of glycoconjugates. This review focuses on recent advances in the technical aspects of cellular glycomic analyses of major classes of glycoconjugates, including N- and O-linked glycans, derived from glycoproteins, proteoglycans and glycosphingolipids. Articles that unveil the glycomics of various biologically important cells, including embryonic and somatic stem cells, induced pluripotent stem (iPS) cells and cancer cells, are discussed.