GZ17-6.02 initiates DNA damage causing autophagosome-dependent HDAC degradation resulting in enhanced anti-PD1 checkpoint inhibitory antibody efficacy

Our studies examined the molecular mechanisms by which the novel cancer therapeutic GZ17-6.02 (NCT03775525) killed GI tumor cells. TZ17-6.02 activated ATM which was responsible for increased phosphorylation of nuclear γH2AX and AMPKα T172. ATM-AMPK signaling was responsible for the subsequent inactivation of mTORC1 and mTORC2, dephosphorylation of ULK1 S757, and increased phosphorylation of ULK1 S317 and of ATG13 S318, which collectively caused enhanced autophagosome formation. GZ17-6.02 interacted with 5-fluorouracil in an additive to greater than additive fashion to kill all of the tested GI tumor cell types. This was associated with greater ATM activation and a greater mammalian target of rapamycin inactivation and autophagosome induction. As a result, autophagy-dependent degradation of multiple histone deacetylase (HDAC) proteins and chaperone proteins occurred. Loss of HDAC expression was causal in reduced expression of programed death ligand 1 (PD-L1), ornithine decarboxylase, and indole amine 2,3-dioxygenase (IDO1) and in the elevated expression of major histocompatibility complex Class IA (MHCA). Treatment with GZ17-6.02 also resulted in enhanced efficacy of a subsequently administered anti-PD1 checkpoint inhibitory antibody. Thus, the primary mode of GZ17-6.02 action is to induce a DNA damage response concomitant with ATM activation, that triggers a series of interconnected molecular events that result in tumor cell death and enhanced immunogenicity.     

Chemotactic G protein-coupled receptors control cell migration by repressing autophagosome biogenesis

Chemotactic migration is a fundamental behavior of cells and its regulation is particularly relevant in physiological processes such as organogenesis and angiogenesis, as well as in pathological processes such as tumor metastasis. The majority of chemotactic stimuli activate cell surface receptors that belong to the G protein-coupled receptor (GPCR) superfamily. Although the autophagy machinery has been shown to play a role in cell migration, its mode of regulation by chemotactic GPCRs remains largely unexplored. We found that ligand-induced activation of 2 chemotactic GPCRs, the chemokine receptor CXCR4 and the urotensin 2 receptor UTS2R, triggers a marked reduction in the biogenesis of autophagosomes, in both HEK-293 and U87 glioblastoma cells. Chemotactic GPCRs exert their anti-autophagic effects through the activation of CAPNs, which prevent the formation of pre-autophagosomal vesicles from the plasma membrane. We further demonstrated that CXCR4- or UTS2R-induced inhibition of autophagy favors the formation of adhesion complexes to the extracellular matrix and is required for chemotactic migration. Altogether, our data reveal a new link between GPCR signaling and the autophagy machinery, and may help to envisage therapeutic strategies in pathological processes such as cancer cell invasion.     

Molecular characterization of a second copy of holocarboxylase synthetase gene (hcs2) in Arabidopsis thaliana.

Holocarboxylase synthetase (HCS), catalyzing the covalent attachment of biotin, is ubiquitously represented in living organisms. Indeed, the biotinylation is a post-translational modification that allows the transformation of inactive biotin-dependent carboxylases, which are committed in fundamental metabolisms such as fatty acid synthesis, into their active holo form. Among other living organisms, plants present a peculiarly complex situation. In pea, HCS activity has been detected in three subcellular compartments and the systematic sequencing of the Arabidopsis genome revealed the occurrence of two hcs genes (hcs1 and hcs2). Hcs1 gene product had been previously characterized at molecular and biochemical levels. Here, by PCR amplification, we cloned an hcs2 cDNA from Arabidopsis thaliana (Ws ecotype) mRNA. We observed the occurrence of multiple cDNA forms which resulted from the alternative splicing of hcs2 mRNA. Furthermore, we evidenced a nucleotide polymorphism at the hcs2 gene within the Ws ecotype, which affected splicing of hcs2 mRNA. This contrasted sharply with the situation at hcs1 locus. However, this polymorphism had no apparent effect on total HCS activity in planta. Finally, hcs2 mRNAs were found 4-fold less abundant than hcs1 mRNA and the most abundant hcs2 mRNA spliced variant should code for a truncated protein. We discuss the possible role of such a multiplicity of putative HCS proteins in plants and discuss the involvement of each of hcs genes in the correct realization of biotinylation.     

Greenhouse Gas Profile of a Plastic Material Derived from a Genetically Modified Plant

Abstract: This article reports an assessment of the global warming potential associated with the life cycle of a biopolymer (poly(hydroxyalkanoate) or PHA) produced in genetically engineered corn developed by Monsanto. The grain corn is harvested in a conventional manner, and the polymer is extracted from the corn stover (i.e., residues such as stalks, leaves and cobs), which would be otherwise left on the field. While corn farming was assessed based on current practice, four different hypothetical PHA production scenarios were tested for the extraction process. Each scenario differed in the energy source used for polymer extraction and compounding, and the results were compared to polyethylene (PE). The first scenario involved burning of the residual biomass (primarily cellulose) remaining after the polymer was extracted from the stover. In the three other scenarios, the use of conventional energy sources of coal, oil, and natural gas were investigated. This study indicates that an integrated system, wherein biomass energy from corn stover provides energy for polymer processing, would result in a better greenhouse gas profile for PHA than for PE. However, plant-based PHA production using fossil fuel sources provides no greenhouse gas advantage over PE, in fact scoring worse than PE. These results are based on a "cradle-to-pellet" modeling as the PHA end-of-life was not quantitatively studied due to complex issues surrounding the actual fate of postconsumer PHA.     

Interannual variability in dissolved inorganic nutrients in northern San Francisco Bay estuary

Nearly two decades of seasonal dissolved inorganic nutrient-salinity distributions in northern San Francisco Bay estuary (1960–1980) illustrate interannual variations in effects of river flow (a nutrient source) and phytoplankton productivity (a nutrient sink). During winter, nutrient sources dominate the nutrient-salinity distribution patterns (nutrients are at or exceed conservative mixing concentrations). During summer, however, the sources and sinks are in close competition. In summers of wet years, the effects of increased river flow often dominate the nutrient distributions (nutrients are at or less than conservative mixing concentrations), whereas in summers of dry years, phytoplankton productivity dominates (the very dry years 1976–1977 were an exception for reasons not yet clearly known). Such source/sink effects also vary with chemical species. During summer the control of phytoplankton on nutrient distributions is apparently strongest for ammonium, less so for nitrate and silica, and is the least for phosphate. Furthermore, the strength of the silica sink (diatom productivity) is at a maximum at intermediate river flows. This relation, which is in agreement with other studies based on phytoplankton abundance and enumeration, is significant to the extent that diatoms are an important food source for herbivores.The balance or lack of balance between nutrient sources and sinks varies from one estuary to another just as it can from one year to another within the same estuary. At one extreme, in some estuaries river flow dominates the estuarine dissolved inorganic nutrient distributions throughout most of the year. At the other extreme, phytoplankton productivity dominates. In northern San Francisco Bay, for example, the phytoplankton nutrient sink is not as strong as in less turbid estuaries. In this estuary, however, river effects, which produce or are associated with near-conservative nutrient distributions, are strong even at flows less than mean-annual flow. Thus, northern San Francisco Bay appears to be an estuary in between the two extremes and is shifted closer to one extreme or the other depending on interannual variations in river flow.     

Exogenous somatostatin raises plasma insulin levels in patients with insulin-dependent diabetes mellitus

The effect of cyclic somatostatin on circulating insulin levels was studied in eight patients with insulin-dependent diabetes mellitus (IDDM). The study was performed after an overnight fast when their subcutaneous depots of insulin had been depleted during i.v. insulin substitution for 18 hours. A constant rate i.v. insulin infusion (0.4 mU/kg/min) was given for 240 min and somatostatin was co-infused between 60-120 min (100 micrograms/h) and 180-240 min (250 micrograms/h) respectively. Plasma insulin, blood glucose and hematocrit were measured at 15 min intervals. Hematocrit fell from 41.7 to 38.3% during the study period. Somatostatin increased the plasma insulin levels, corrected for the changes of hematocrit, by approximately 8% in the low dose (P less than 0.05) as well as in the high dose (P less than 0.05) period. It is concluded that somatostatin interferes with the clearance of insulin thereby increasing the circulating plasma insulin levels in IDDM patients without residual insulin secretion.     

The apocytochrome b gene of Chlamydomonas smithii contains a mobile intron related to both Saccharomyces and Neurospora introns.

Summary The mitochondrial DNA of the two interfertile algal species Chlamydomonas smithii and Chlamydomonas reinhardtii are co-linear with the exception of ca. 1 kb insertion (the a insert) present in C. smithii DNA only. In vegetative diploids resulting from interspecific crosses, mitochondrial genomes are transmitted biparentally except for the a insert which is transmitted to all C. reinhardtii molecules in a manner reminiscent of the intron-mediated conversion event that occurs at the omega locus in yeast mitochondria, under the action of the I-SceI endonuclease. Here we report that the insert corresponds to a typical group I intron of 1075 bp, inserted within the gene for apocytochrome b and containing a 237 codon open reading frame (ORF). We also report the complete sequence of the apocytochrome b gene of C. smithii. Comparison with the sequence of the same gene in C. reinhardtii reveals the precise intron insertion site. These data, together with the previous genetic data provide the first example of intron mobility in mitochondria of the plant kingdom. The product of the intronic ORF shows 36% amino acid identity with the I-SceI endonuclease whereas the intron ribozyme shows a 60% identity at the nucleotide level with the Neurospora crassa cob · 1 intron. The possibility of a recent horizontal transfer of introns between fungi and algae is discussed.     

Biochemical,genetic and molecular characterization of new respiratory-deficient mutants inChlamydomonas reinhardtii

Eight respiratory-deficient mutants ofChlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternalmt ^- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochromec oxido-reductase) and complex IV (cytochromec oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochromeb (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses.Anin vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.