Protein kinase Cepsilon is important for migration of neuroblastoma cells

<p>Abstract</p> <p>Background</p> <p>Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility.</p> <p>Methods</p> <p>PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot.</p> <p>Results</p> <p>Stimulation with 12-<it>O</it>-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS.</p> <p>Conclusion</p> <p>PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration.</p

Synthesis and characterization of ZnO nanostructures using palm olein as biotemplate

Background: A green approach to synthesize nanomaterials using biotemplates has been subjected to intense research due to several advantages. Palm olein as a biotemplate offers the benefits of eco-friendliness, low-cost and scale-up for large scale production. Therefore, the effect of palm olein on morphology and surface properties of ZnO nanostructures were investigated. Results: The results indicate that palm olein as a biotemplate can be used to modify the shape and size of ZnO particles synthesized by hydrothermal method. Different morphology including flake-, flower- and three dimensional star-like structures were obtained. FTIR study indicated the reaction between carboxyl group of palm olein and zinc species had taken place. Specific surface area enhanced while no considerable change were observed in optical properties. Conclusion: Phase-pure ZnO particles were successfully synthesized using palm olein as soft biotemplating agent by hydrothermal method. The physico-chemical properties of the resulting ZnO particles can be tuned using the ratio of palm olein to Zn cation

A Systematic Screen for Tube Morphogenesis and Branching Genes in the Drosophila Tracheal System

Many signaling proteins and transcription factors that induce and pattern organs have been identified, but relatively few of the downstream effectors that execute morphogenesis programs. Because such morphogenesis genes may function in many organs and developmental processes, mutations in them are expected to be pleiotropic and hence ignored or discarded in most standard genetic screens. Here we describe a systematic screen designed to identify all Drosophila third chromosome genes (∼40% of the genome) that function in development of the tracheal system, a tubular respiratory organ that provides a paradigm for branching morphogenesis. To identify potentially pleiotropic morphogenesis genes, the screen included analysis of marked clones of homozygous mutant tracheal cells in heterozygous animals, plus a secondary screen to exclude mutations in general “house-keeping” genes. From a collection including more than 5,000 lethal mutations, we identified 133 mutations representing ∼70 or more genes that subdivide the tracheal terminal branching program into six genetically separable steps, a previously established cell specification step plus five major morphogenesis and maturation steps: branching, growth, tubulogenesis, gas-filling, and maintenance. Molecular identification of 14 of the 70 genes demonstrates that they include six previously known tracheal genes, each with a novel function revealed by clonal analysis, and two well-known growth suppressors that establish an integral role for cell growth control in branching morphogenesis. The rest are new tracheal genes that function in morphogenesis and maturation, many through cytoskeletal and secretory pathways. The results suggest systematic genetic screens that include clonal analysis can elucidate the full organogenesis program and that over 200 patterning and morphogenesis genes are required to build even a relatively simple organ such as the Drosophila tracheal system

Differential roles of the Drosophila EMT-inducing transcription factors Snail and Serpent in driving primary tumour growth.

Several transcription factors have been identified that activate an epithelial-to-mesenchymal transition (EMT), which endows cells with the capacity to break through basement membranes and migrate away from their site of origin. A key program in development, in recent years it has been shown to be a crucial driver of tumour invasion and metastasis. However, several of these EMT-inducing transcription factors are often expressed long before the initiation of the invasion-metastasis cascade as well as in non-invasive tumours. Increasing evidence suggests that they may promote primary tumour growth, but their precise role in this process remains to be elucidated. To investigate this issue we have focused our studies on two Drosophila transcription factors, the classic EMT inducer Snail and the Drosophila orthologue of hGATAs4/6, Serpent, which drives an alternative mechanism of EMT; both Snail and GATA are specifically expressed in a number of human cancers, particularly at the invasive front and in metastasis. Thus, we recreated conditions of Snail and of Serpent high expression in the fly imaginal wing disc and analysed their effect. While either Snail or Serpent induced a profound loss of epithelial polarity and tissue organisation, Serpent but not Snail also induced an increase in the size of wing discs. Furthermore, the Serpent-induced tumour-like tissues were able to grow extensively when transplanted into the abdomen of adult hosts. We found the differences between Snail and Serpent to correlate with the genetic program they elicit; while activation of either results in an increase in the expression of Yorki target genes, Serpent additionally activates the Ras signalling pathway. These results provide insight into how transcription factors that induce EMT can also promote primary tumour growth, and how in some cases such as GATA factors a ‘multi hit’ effect may be achieved through the aberrant activation of just a single gene

Genome-Wide Analysis Reveals a Major Role in Cell Fate Maintenance and an Unexpected Role in Endoreduplication for the Drosophila FoxA Gene Fork Head

Transcription factors drive organogenesis, from the initiation of cell fate decisions to the maintenance and implementation of these decisions. The Drosophila embryonic salivary gland provides an excellent platform for unraveling the underlying transcriptional networks of organ development because Drosophila is relatively unencumbered by significant genetic redundancy. The highly conserved FoxA family transcription factors are essential for various aspects of organogenesis in all animals that have been studied. Here, we explore the role of the single Drosophila FoxA protein Fork head (Fkh) in salivary gland organogenesis using two genome-wide strategies. A large-scale in situ hybridization analysis reveals a major role for Fkh in maintaining the salivary gland fate decision and controlling salivary gland physiological activity, in addition to its previously known roles in morphogenesis and survival. The majority of salivary gland genes (59%) are affected by fkh loss, mainly at later stages of salivary gland development. We show that global expression of Fkh cannot drive ectopic salivary gland formation. Thus, unlike the worm FoxA protein PHA-4, Fkh does not function to specify cell fate. In addition, Fkh only indirectly regulates many salivary gland genes, which is also distinct from the role of PHA-4 in organogenesis. Our microarray analyses reveal unexpected roles for Fkh in blocking terminal differentiation and in endoreduplication in the salivary gland and in other Fkh-expressing embryonic tissues. Overall, this study demonstrates an important role for Fkh in determining how an organ preserves its identity throughout development and provides an alternative paradigm for how FoxA proteins function in organogenesis

Detection of genotype-1 of dengue virus serotype 3 for the first time and complete genome analysis of dengue viruses during the 2018 epidemic in Mandalay, Upper Myanmar

Background: Dengue (DEN) is a neglected tropical disease, and surveillance of dengue virus (DENV) serotypes and genotypes is critical for the early detection of outbreaks. Risk factors for outbreaks include the emergence of new genotypes and serotype shifting.Methodology and principal findings: To understand the genomic and viral characteristics of DENV-infected patients, we conducted a cross-sectional descriptive study among pediatric patients admitted at the 550-bedded Mandalay Children Hospital during the 2018 DEN endemic season. We conducted virus isolation, serological tests, viremia level measurement, and whole-genome sequencing. Among the 202 serum samples, we detected 85 samples with DENV (46 DENV-1, 10 DENV-3, 26 DENV-4 and three multiple serotype co-infections) via reverse transcription quantitative/real-time PCR (RT-qPCR), and we obtained 49 DENV isolates (31 DENV-1, 10 DENV-3 and 8 DEN-4). We did not detect DENV-2 in this study. The viral genome levels in serum did not differ significantly among virus serotypes, infection status (primary versus secondary) and disease severity. Based on the phylogenetic analysis, we identified DENV-1 genotype-1, DENV-4 genotype-1 and DENV-3 genotype-3 and genotype-1 which was detected for the first time. Next-generation sequencing analysis revealed greater frequencies of nonsynonymous and synonymous mutations per gene in the nonstructural genes. Moreover, mutation rates were also higher among DENV-1.Conclusion/Significance: In conclusion, there was an increasing trend of DENV-3 cases during DENV endemic season in 2018 with the first detection of the genotype 1. However, DENV-1 has remained the predominant serotype in this study area since 2013, and we identified stop codon mutations in the DENV-1 genome. This report is the first to feature a complete genome analysis of the strains of DENV-3 and DENV-4 circulating among pediatric patients in Myanmar. This study highlighted the importance of annual surveillance for a better understanding of the molecular epidemiology of DENVs

Epithelial tube morphogenesis during Drosophila tracheal development requires Piopio, a luminal ZP protein

The formation of branched epithelial networks is fundamental to the development of many organs, such as the lung, the kidney or the vasculature. Little is known about the mechanisms that control cell rearrangements during tubulogenesis and regulate the size of individual tubes. Recent studies indicate that whereas the basal surface of tube cells interacts with the surrounding tissues and helps to shape the ramification pattern of tubular organs, the apical surface has an important role in the regulation of tube diameter and tube growth. Here we report that two proteins, Piopio (Pio) and Dumpy (Dp), containing a zona pellucida (ZP) domain are essential for the generation of the interconnected tracheal network in Drosophila melanogaster. Pio is secreted apically, accumulates in the tracheal lumen and possibly interacts with Dp through the ZP domains. In the absence of Pio and Dp, multicellular tubes do not rearrange through cell elongation and cell intercalation to form narrow tubes with autocellular junctions; instead they are transformed into multicellular cysts, which leads to a severe disruption of the branched pattern. We propose that an extracellular matrix containing Pio and Dp provides a structural network in the luminal space, around which cell rearrangements can take place in an ordered fashion without losing interconnections. Our results suggest that a similar structural role might be attributed to other ZP-domain proteins in the formation of different branched organs