Translational Validation of Personalized Treatment Strategy Based on Genetic Characteristics of Glioblastoma

Glioblastoma (GBM) heterogeneity in the genomic and phenotypic properties has potentiated personalized approach against specific therapeutic targets of each GBM patient. The Cancer Genome Atlas (TCGA) Research Network has been established the comprehensive genomic abnormalities of GBM, which sub-classified GBMs into 4 different molecular subtypes. The molecular subtypes could be utilized to develop personalized treatment strategy for each subtype. We applied a classifying method, NTP (Nearest Template Prediction) method to determine molecular subtype of each GBM patient and corresponding orthotopic xenograft animal model. The models were derived from GBM cells dissociated from patient''s surgical sample. Specific drug candidates for each subtype were selected using an integrated pharmacological network database (PharmDB), which link drugs with subtype specific genes. Treatment effects of the drug candidates were determined by in vitro limiting dilution assay using patient-derived GBM cells primarily cultured from orthotopic xenograft tumors. The consistent identification of molecular subtype by the NTP method was validated using TCGA database. When subtypes were determined by the NTP method, orthotopic xenograft animal models faithfully maintained the molecular subtypes of parental tumors. Subtype specific drugs not only showed significant inhibition effects on the in vitro clonogenicity of patient-derived GBM cells but also synergistically reversed temozolomide resistance of MGMT-unmethylated patient-derived GBM cells. However, inhibitory effects on the clonogenicity were not totally subtype-specific. Personalized treatment approach based on genetic characteristics of each GBM could make better treatment outcomes of GBMs, although more sophisticated classifying techniques and subtype specific drugs need to be further elucidated.     

Suppression of Cpn10 Increases Mitochondrial Fission and Dysfunction in Neuroblastoma Cells

To date, several regulatory proteins involved in mitochondrial dynamics have been identified. However, the precise mechanism coordinating these complex processes remains unclear. Mitochondrial chaperones regulate mitochondrial function and structure. Chaperonin 10 (Cpn10) interacts with heat shock protein 60 (HSP60) and functions as a co-chaperone. In this study, we found that down-regulation of Cpn10 highly promoted mitochondrial fragmentation in SK-N-MC and SH-SY5Y neuroblastoma cells. Both genetic and chemical inhibition of Drp1 suppressed the mitochondrial fragmentation induced by Cpn10 reduction. Reactive oxygen species (ROS) generation in 3-NP-treated cells was markedly enhanced by Cpn10 knock down. Depletion of Cpn10 synergistically increased cell death in response to 3-NP treatment. Furthermore, inhibition of Drp1 recovered Cpn10-mediated mitochondrial dysfunction in 3-NP-treated cells. Moreover, an ROS scavenger suppressed cell death mediated by Cpn10 knockdown in 3-NP-treated cells. Taken together, these results showed that down-regulation of Cpn10 increased mitochondrial fragmentation and potentiated 3-NP-mediated mitochondrial dysfunction in neuroblastoma cells.     

Retinal pigment epithelial cells undergoing mitotic catastrophe are vulnerable to autophagy inhibition

The increased mitochondrial DNA damage leads to altered functional capacities of retinal pigment epithelial (RPE) cells. A previous study showed the increased autophagy in RPE cells caused by low concentrations of rotenone, a selective inhibitor of mitochondrial complex I. However, the mechanism by which autophagy regulates RPE cell death is still unclear. In the present study, we examined the mechanism underlying the regulation of RPE cell death through the inhibition of mitochondrial complex I. We report herein that rotenone induced mitotic catastrophe (MC) in RPE cells. We further observed an increased level of autophagy in the RPE cells undergoing MC (RPE-MC cells). Importantly, autophagy inhibition induced nonapoptotic cell death in RPE-MC cells. These findings indicate that autophagy has a pivotal role in the survival of RPE-MC cells. We next observed PINK1 accumulation in the mitochondrial membrane and parkin translocation into the mitochondria from the cytosol in the rotenone-treated RPE-MC cells, which indicates that increased mitophagy accompanies MC in ARPE-19 cells. Noticeably, the mitophagy also contributed to the cytoprotection of RPE-MC cells. Although there might be a significant gap in the roles of autophagy and mitophagy in the RPE cells in vivo, our in vitro study suggests that autophagy and mitophagy presumably prevent the RPE-MC cells from plunging into cell death, resulting in the prevention of RPE cell loss.Cell death is a process that is both complementary and antagonistic to cell division in order to maintain tissue homeostasis, and cell death has a pivotal role in several physiological processes and diseases.¹ The most extensively studied category, apoptosis, is characterized by the massive activation of caspases, chromatin condensation, and a reduction in cell volume. Necrosis is characterized by an increase in cell volume, the swelling of organelles, and the rupture of the plasma membrane and is largely considered an accidental, uncontrolled type of cell death.² Necroptosis is a regulated necrotic cell death that is triggered by broad caspase inhibition in the presence of death receptor ligands and is characterized by necrotic cell death morphology. Autophagy is a degradative lysosomal pathway that is characterized by the accumulation of cytoplasmic material in the vacuoles for bulk degradation by lysosomal enzymes. Although autophagy has a pivotal role in cell survival, increased autophagic activity is often associated with cell death.² Mitotic catastrophe (MC) is a type of cell death that results from a failure to undergo mitosis after DNA damage, leading to tetraploidy or endopolyploidy. Cells undergoing MC usually form large cells with multiple micronuclei.³Retinal pigment epithelial (RPE) cells form a single layer of cells adjacent to the photoreceptor outer segment (POS) of the retina, and these cells have pivotal roles in the maintenance of the POS cells. RPE cell death is a significant factor in several ocular pathological conditions, such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). AMD is a progressive degeneration of the macula and is broadly classified as either dry or wet. The dry form of AMD is more common and is characterized by the presence of drusen in the macula. Mitochondrial DNA variants of respiratory complex I are associated with an increased risk of AMD.⁴ Because damage to and the death of RPEs are crucial and perhaps even triggering events in AMD,⁵ protection against RPE cell death could delay the onset of AMD. Conversely, RPE cells significantly contribute to the formation of the epiretinal membrane in PVR. Thus, the induction of RPE cell death in the epiretinal membranes could be a new approach to inhibit cellular proliferation in PVR.⁶ Most studies concerning RPE cell death in the context of these ocular pathological conditions have focused on two types of cell death, apoptosis and necrosis.Although advances have been made in the understanding of RPE cell death, there is little information concerning the role of autophagy in the RPE cell death associated with these ocular pathological conditions. Each day, RPE cells phagocytose and digest the distal parts of the POS, which are ultimately degraded in the lysosomes.^{7, 8, 9} The interplay of phagocytosis and autophagy within the RPE is required for both POS degradation and the maintenance of retinoid levels to support vision.⁹ In the RPE cells of old eyes, this physiological lysosomal load may be further increased to remove damaged material, and insufficient digestion of the damaged macromolecules and organelles by old RPE cells will lead to progressive accumulation of biological ‘garbage'', such as lipofuscin.¹⁰ Thus, abnormalities in the lysosome-dependent degradation of shed POS debris can contribute to the degeneration of RPE cells. A previous study suggested that age-related changes in autophagy may underlie the genetic susceptibility found in AMD patients and may be associated with the pathogenesis of AMD.¹⁰ However, the mechanism by which autophagy regulates RPE cell demise in AMD is still unclear. The role of autophagy in the proliferation of the RPE cells in PVR and its regulation as a therapeutic strategy for PVR have not been documented yet.Rotenone, a natural isoflavonoid produced by plants, is a selective and stoichiometric inhibitor of mitochondrial complex I.¹¹ More specifically, rotenone blocks NADH oxidation by the NADH-ubiquinone oxide reductase enzymatic complex, which results in the inhibition of mitochondrial respiration and a reduction in ATP synthesis.^{12, 13, 14} Rotenone treatment also results in the production of reactive oxygen species (ROS), eventually leading to cell death.^{15, 16} Several studies have shown that rotenone causes an accumulation of autophagic vacuoles, perhaps in response to the inhibition of mitochondrial function and the generation of oxidative stress.^{17, 18, 19} Irrespective of that activity of rotenone has been lively studied in various cells, the effect of rotenone on RPE cells has rarely been studied. A previous study using an in vitro system revealed that low concentrations of rotenone resulted in mtDNA damage in RPE cells and suggested that the increased autophagy caused by rotenone treatment in aged RPE cells could affect the formation of drusen and AMD.¹⁰ However, the mechanism by which rotenone regulates RPE cell demise remains unclear.We undertook this study to elucidate the mechanism regulating the demise of RPE cells that are damaged by mitochondrial complex I inhibition. We report herein that rotenone induces MC in RPE cells. Additionally, we show that RPE cells undergoing mitotic catastrophe (RPE-MC cells) induced by mitochondrial complex I inhibition are vulnerable to autophagy inhibition.     

Secretion of a Truncated Osteopetrosis-associated Transmembrane Protein 1 (OSTM1) Mutant Inhibits Osteoclastogenesis through Down-regulation of the B Lymphocyte-induced Maturation Protein 1 (BLIMP1)-Nuclear Factor of Activated T Cells c1 (NFATc1) Axis

Genetic mutations in osteoclastogenic genes are closely associated with osteopetrotic bone diseases. Genetic defects in OSTM1 (osteopetrosis-associated transmembrane protein 1) cause autosomal recessive osteopetrosis in humans. In particular, OSTM1 mutations that exclude the transmembrane domain might lead to the production of a secreted form of truncated OSTM1. However, the precise role of the secreted form of truncated OSTM1 remains unknown. In this study, we analyzed the functional role of truncated OSTM1 in osteoclastogenesis. Here, we showed that a secreted form of truncated OSTM1 binds to the cell surface of osteoclast (OC) precursors and inhibits the formation of multinucleated OCs through the reduction of cell fusion and survival. Truncated OSTM1 significantly inhibited the expression of OC marker genes through the down-regulation of the BLIMP1 (B lymphocyte-induced maturation protein 1)-NFATc1 (nuclear factor of activated T cells c1) axis. Finally, we demonstrated that truncated OSTM1 reduces lipopolysaccharide-induced bone destruction in vivo. Thus, these findings suggest that autosomal recessive osteopetrosis patients with an OSTM1 gene mutation lacking the transmembrane domain produce a secreted form of truncated OSTM1 that inhibits osteoclastogenesis.     

Genetic organization and conjugal plasmid DNA transfer of pHP69, a plasmid from a Korean isolate of Helicobacter pylori

We isolated pHP69, a 9,153 bp plasmid from Helicobacter pylori with a 33.98% (G+C) content. We identified 11 open reading frames (ORFs), including replication initiation protein A (repA), fic (cAMP-induced filamentation protein), mccC, mccB, mobA, mobD, mobB, and mobC, as well as four 22 bp tandem repeat sequences. The nucleic acid and predicted amino acid sequences of these ORFs exhibited significant homology to those of other H. pylori plasmids. pHP69 repA encodes a replication initiation protein and its amino acid sequence is similar to those of replicase proteins from theta-type plasmids. pHP69 contains two types of repeat sequences (R1 and R2), a MOBHEN family mobilization region comprising mobC, mobA, mobB, and mobD, and genes encoding microcin B and C. Among the 36 H. pylori strains containing plasmids, mobA or mccBC are present in 12 or 6, respectively and 3 contain both genes. To examine intrinsic capability of H. pylori for conjugative plasmid transfer, a shuttle vector pBHP69KH containing pHP69 and replication origin of pBR322 was constructed. It was shown that this vector could stably replicate and be mobilized among clinical H. pylori strains and demonstrated to gene transfer by natural plasmid.     

Complete Genome Analysis of Three Live Attenuated Rinderpest Virus Vaccine Strains Derived through Serial Passages in Different Culture Systems

The genomes of three South Korean Rinderpest virus vaccine strains (L72, LA77, and LA96) were analyzed in order to investigate their genetic variability. These three vaccine strains were all derived from the same virus strain origin (Fusan) through repeated passages in different culture systems. The full genome length of the three strains was 15,882 nucleotides, and the sequence similarity between the three South Korean RPV strains at the nucleotide level was 98.1 to 98.9%. The genetic distance between Nakamura III, L72, LA77, LA96, and LATC06 and the Kabete strain was greater than that between the Fusan and Kabete strains for the P, V, and C genes. The difference in pathogenicity among these strains might be due to the V gene, which has a positive (>1) selection ratio based on the analysis of synonymous (dS) and nonsynonymous (dN) substitution rates (dN/dS ratio [ω]).     

Bioproduction of lauryl lactone and 4-vinyl guaiacol as value-added chemicals in two-phase biotransformation systems

Recombinant Escherichia coli whole-cell biocatalysts harboring either a Baeyer–Villiger monooxygenase or ferulic acid decarboxylase were employed in organic-aqueous two-phase bioreactor systems. The feasibility of the bioproduction of water-insoluble products, viz., lauryl lactone from cyclododecanone and 4-vinyl guaiacol from ferulic acid were examined. Using hexadecane as the organic phase, 10∼16 g of lauryl lactone were produced in a 3-l bioreactor that operated in a semicontinuous mode compared to 2.4 g of product in a batch mode. For the decarboxylation of ferulic acid, a new recombinant biocatalyst, ferulic acid decarboxylase derived from Bacillus pumilus, was constructed. Selected solvents as well as other parameters for in situ recovery of vinyl guaiacol were investigated. Up to 13.8 g vinyl guaiacol (purity of 98.4%) were obtained from 25 g of ferulic acid in a 2-l working volume bioreactor by using octane as organic phase. These selected examples highlight the superiority of the two-phase biotransformations systems over the conventional batch mode.     

Influences of Blue and Red Light on the Photosynthetic Apparatus of Chlorella kessleri*

In white light of 33.2 μmol . m^?2 . s^?1 oxygen evolution of Chlorella kessleri is about 30 % higher after growth in blue light than after growth in red light of the same quantum fluence rate. When determined by the light-induced absorbance change at γ 820 nm, blue light-adapted cells possess about 60% more reaction centres per total chlorophyll in photosystem II. Correspondingly, the cells exhibit about 30% more Hill activity of PS II. Conversely, red light-adapted cells contain relatively more reaction centres and higher electron flow capacities of photosystem I. The distribution of total chlorophyll among the pigment-protein complexes, CPI, CPIa, CPa, and LHC II, corresponds to these data. There is more chlorophyll associated with the light-harvesting complex of PS II, LHC II, in cells under blue light conditions, but more chlorophyll bound to both complexes of PS I, CPI and CPIa, in cells under red light conditions. The respective ratios of chlorophyll a/chlorophyll b of all complexes are identical for blue and red light-adapted cells. This results in a higher relative amount of chlorophyll b in blue light-adapted cells. Total carotenoids per total chlorophyll are increased by 20% in red light-adapted cells. Their distribution among the pigment-protein complexes is unknown, however the ratios of lutein, neoxanthin and violaxanthin extractable from LHC II are different in blue (32.1:35.9:32.0) and in red (51.4:26.7:21.9) light-adaptod cells.