eIF5A Promotes Translation Elongation, Polysome Disassembly and Stress Granule Assembly

Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate in cells exposed to environmental stress. We have identified ornithine decarboxylase (ODC), an enzyme required for polyamine synthesis, and eIF5A, a polyamine (hypusine)-modified translation factor, as proteins required for arsenite-induced SG assembly. Knockdown of deoxyhypusine synthase (DHS) or treatment with a deoxyhypusine synthase inhibitor (GC7) prevents hypusine modification of eIF5A as well as arsenite-induced polysome disassembly and stress granule assembly. Time-course analysis reveals that this is due to a slowing of stress-induced ribosome run-off in cells lacking hypusine-eIF5A. Whereas eIF5A only marginally affects protein synthesis under normal conditions, it is required for the rapid onset of stress-induced translational repression. Our results reveal that hypusine-eIF5A-facilitated translation elongation promotes arsenite-induced polysome disassembly and stress granule assembly in cells subjected to adverse environmental conditions

GWAS and meta-analysis identifies 49 genetic variants underlying critical COVID-19

Critical illness in COVID-19 is an extreme and clinically homogeneous disease phenotype that we have previously shown to be highly efficient for discovery of genetic associations. Despite the advanced stage of illness at presentation, we have shown that host genetics in patients who are critically ill with COVID-19 can identify immunomodulatory therapies with strong beneficial effects in this group. Here we analyse 24,202 cases of COVID-19 with critical illness comprising a combination of microarray genotype and whole-genome sequencing data from cases of critical illness in the international GenOMICC (11,440 cases) study, combined with other studies recruiting hospitalized patients with a strong focus on severe and critical disease: ISARIC4C (676 cases) and the SCOURGE consortium (5,934 cases). To put these results in the context of existing work, we conduct a meta-analysis of the new GenOMICC genome-wide association study (GWAS) results with previously published data. We find 49 genome-wide significant associations, of which 16 have not been reported previously. To investigate the therapeutic implications of these findings, we infer the structural consequences of protein-coding variants, and combine our GWAS results with gene expression data using a monocyte transcriptome-wide association study (TWAS) model, as well as gene and protein expression using Mendelian randomization. We identify potentially druggable targets in multiple systems, including inflammatory signalling (JAK1), monocyte–macrophage activation and endothelial permeability (PDE4A), immunometabolism (SLC2A5 and AK5), and host factors required for viral entry and replication (TMPRSS2 and RAB2A)

GWAS and meta-analysis identifies 49 genetic variants underlying critical COVID-19

Critical illness in COVID-19 is an extreme and clinically homogeneous disease phenotype that we have previously shown to be highly efficient for discovery of genetic associations. Despite the advanced stage of illness at presentation, we have shown that host genetics in patients who are critically ill with COVID-19 can identify immunomodulatory therapies with strong beneficial effects in this group. Here we analyse 24,202 cases of COVID-19 with critical illness comprising a combination of microarray genotype and whole-genome sequencing data from cases of critical illness in the international GenOMICC (11,440 cases) study, combined with other studies recruiting hospitalized patients with a strong focus on severe and critical disease: ISARIC4C (676 cases) and the SCOURGE consortium (5,934 cases). To put these results in the context of existing work, we conduct a meta-analysis of the new GenOMICC genome-wide association study (GWAS) results with previously published data. We find 49 genome-wide significant associations, of which 16 have not been reported previously. To investigate the therapeutic implications of these findings, we infer the structural consequences of protein-coding variants, and combine our GWAS results with gene expression data using a monocyte transcriptome-wide association study (TWAS) model, as well as gene and protein expression using Mendelian randomization. We identify potentially druggable targets in multiple systems, including inflammatory signalling (JAK1), monocyte–macrophage activation and endothelial permeability (PDE4A), immunometabolism (SLC2A5 and AK5), and host factors required for viral entry and replication (TMPRSS2 and RAB2A)

A Genome-wide Screen for Neurospora crassa Transcription Factors Regulating Glycogen Metabolism

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Transcription factors play a key role in transcription regulation as they recognize and directly bind to defined sites in promoter regions of target genes, and thus modulate differential expression. The overall process is extremely dynamic, as they have to move through the nucleus and transiently bind to chromatin in order to regulate gene transcription. To identify transcription factors that affect glycogen accumulation in Neurospora crassa, we performed a systematic screen of a deletion strains set generated by the Neurospora Knockout Project and available at the Fungal Genetics Stock Center. In a wild-type strain of N. crassa, glycogen content reaches a maximal level at the end of the exponential growth phase, but upon heat stress the glycogen content rapidly drops. The gene encoding glycogen synthase (gsn) is transcriptionally down-regulated when the mycelium is exposed to the same stress condition. We identified 17 deleted strains having glycogen accumulation profiles different from that of the wild-type strain under both normal growth and heat stress conditions. Most of the transcription factors identified were annotated as hypothetical protein, however some of them, such as the PacC, XlnR, and NIT2 proteins, were biochemically well-characterized either in N. crassa or in other fungi. The identification of some of the transcription factors was coincident with the presence of DNA-binding motifs specific for the transcription factors in the gsn 5'-flanking region, and some of these DNA-binding motifs were demonstrated to be functional by Electrophoretic Mobility Shift Assay (EMSA) experiments. Strains knocked-out in these transcription factors presented impairment in the regulation of gsn expression, suggesting that the transcription factors regulate glycogen accumulation by directly regulating gsn gene expression. Five selected mutant strains showed defects in cell cycle progression, and two transcription factors were light-regulated. The results indicate that there are connections linking different cellular processes, such as metabolism control, biological clock, and cell cycle progression. Molecular & Cellular Proteomics 10: 10.1074/mcp.M111.007963, 1-13, 2011.1011Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Expression of correctly processed human growth hormone in seeds of transgenic tobacco plants

Human growth hormone was expressed in transgenic tobacco seeds using the monocot tissue-specific promoter from sorghum gamma-kafirin seed storage protein gene. During tobacco seed ripening, the expressed hormone was directed to the endoplasmic reticulum by a signal peptide from a Coix prolamin and was secreted into the apoplastic space, where it accounted for 0.16% of total soluble seed protein. The expressed hormone has the same amino acid sequence and receptor-binding properties as the native mature hormone.61475

Effect of lipid infusion on metabolism and force of rat skeletal muscles during intense contractions

The hypothesis that during intense muscle contraction induced by electrical stimulation, long chain fatty acids (LCFA) might reduce mitochondrial ATP/ADP ratio, raising the contribution of glycolysis for ATP production was examined. The effect of a lipid infusion (Lipovenus emulsion) on UCP-3 mRNA level, lactate, glucose-6-phosphate (G-6P) and glycogen content was investigated in rat. Blood samples for determination of free fatty acids and lactate were collected at 0, 30 and 60 min during rest and at 0, 10 and 20 min during muscle contraction. The content of lactate, glycogen and G-6P was also determined in soleus (SO), red gastrocnemius (RG) and white gastrocnemius (WG) muscles collected immediately after muscle contraction period. In addition, the force level was determined during muscle contractions. The effect of Lipovenus emulsion on respiration of mitochondria isolated from rat skeletal muscle, and content of UCP-3 and lactate in cultured skeletal muscle cells was also determined. The in vivo experiments showed that Lipovenus induced a significant increase of UCP-3 mRNA levels. After Lipovenus infusion, lactate level was increased in RG muscle only, whereas the contents of glycogen and G-6P were decreased in both RG and WG muscles (P 0.05). The in vitro experiments showed that Lipovenus infusion induced a significant increase in mitochondrial respiration, but had no effect on UCP-3 content. Lactate concentration was significantly increased in the culture medium of stimulated cells in the control and Lipovenus groups compared with the respective not-stimulated cells (P < 0.05). We concluded that as mitochondrial function becomes limited by the FFA-uncoupling effect, the ATP demand is mainly supplied by anaerobic glucose metabolism preventing an expected decrease in muscle contraction performance.204173021322