Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: Few components of the TORC1 pathway remain in the P. falciparum genome. (A) A simplified representation of the major TOR complex 1 ( TORC1 ) components, regulators, targets, and converging pathways across several eukaryote lineages. Solid circles (●) indicate the presence of the gene coding for the component in representative members of the lineage, and hollow circles (O) indicate the absence of the corresponding component in the lineage. (B) An illustration of the generalized animal PI3K-TORC1 signaling cascade used to regulate cellular growth in the presence of amino acids and other growth factors (left) and a projection of this pathway in Plasmodium spp. based on the conserved components (right). Plasmodium spp. lack a class I PI3K enzyme and the other components ( PTEN , PDK , an Akt homolog containing a PH domain) typically associated with this signaling cascade. Plasmodium spp. do encode a class III PI3K enzyme (PF3D7_0515300) whose ortholog has been implicated in TORC1 signaling in human cells, as well as a PH domain lacking PKB family kinase (PF3D7_1246900) resembling human S6K . The genomes of Plasmodium parasites also encode an apparent ortholog of the TORC1 -dependent RNA polymerase III regulator Maf1 (PF3D7_0416500).

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques:

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: Functional complementation of Maf1-knockout yeast cells with a chimeric P. falciparum Maf1. (A) Schematics showing the key structural features of the yeast and Plasmodium Maf1 orthologs (Nt, N terminus; Ct, C terminus; NLS, nuclear localization signal). Vertical lines indicate the homologous region exchanged to generate the chimera for complementation. *P, known site of phosphorylation in the yeast protein. (B) Fivefold serial dilutions of from ~5,000 to ~8 yeast cells transformed with the indicated complementation vectors were plated on normal rich media or on rich media supplemented with 10 nM sirolimus, which is lethal to Maf1 knockout yeast cells. Images are representative of one of three biological replicates.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Functional Assay, Knock-Out, Phospho-proteomics, Transformation Assay

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: The PB-11 mutant carries a piggyBac insertion in the 5′UTR of the Maf1 gene. (A) Schematics of the Maf1 genomic locus and piggyBac insertion in wild-type and mutant parasites. Numbers below the chromosomal line indicate the reported and verified transposon insertion positions relative to the Maf1 start codon. Right-angled arrows and numbers above the chromosome line indicate the transcription start sites in wild-type (−93 nt) and PB-11 mutant (−395 nt) cells as determined by 5′-RACE. The Maf1 transcript in the PB-11 mutant arises from within the piggyBac transposon and is likely due to bidirectional promoter activity of the calmodulin promoter fragment used for drug selection in the transposon. (B) The upstream insertion site was confirmed by whole-genome sequencing (top). Coverage depth plots indicate the total number of reads mapping to each base pair around the two termini of the piggyBac insertion at the −53 TTAA site upstream of the Maf1 start codon. The maximum read coverage for each end of the transposon is indicated by the axes on the left and right (bottom). Raw reads aligned to the termini of the transposon surrounding the −53 insertion site. Each read is 100 bp in length. Colored spots indicate base mismatches within a given read and the genomic sequence.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Activity Assay, Selection, Sequencing

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: The PB-11 mutant displays an abnormal Maf1 mRNA expression profile. An analysis of the time course of Maf1 mRNA expression was performed using qRT-PCR at seven time points across the intraerythrocytic cycle and synchronous wild-type (NF54) and mutant (PB-11) parasites. Maf1 expression was quantified relative to that of the seryl-tRNA ligase transcript (PF3D7_0717700). Points represent individual biological replicates (three in total), and curves represent LOESS smoothed models fitted to the data, with the 95% confidence interval indicated by shading. ΔΔCT, threshold cycle method; hpi, hour post-red blood cell (RBC) invasion.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: Maf1 mutant parasites cannot recover from a prolonged dormancy-like state induced by isoleucine starvation. Synchronous young ring-stage parasites (approximately 4 h postinvasion) were washed repeatedly and transferred to medium lacking isoleucine (Ile) for the indicated times. Recovery data denote transfer back to normal culture medium (containing isoleucine) for 72 h of growth. Parasitemia was quantified by flow cytometry. Growth was measured relative to the final level of parasitemia of a control culture incubated in normal culture medium for 72 h. P values were calculated using t tests of three biological replicates.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Flow Cytometry, Control, Incubation

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: The PB-11 mutant remains viable but loses the ability to recover within the first 72 h of starvation. (A) Parasitemia of Maf1 mutant parasites decreases more rapidly during prolonged isoleucine starvation. Synchronous young ring-stage NF54 (WT) and Maf1 insertion mutant (PB-11) parasites were washed repeatedly and transferred to medium lacking isoleucine. Samples of each parasite line were taken at 8-h intervals over the course of a 9-day (216-h) period, and parasitemia was determined by flow cytometry. Lines represent LOESS curves fitted to the data for each of three biological replicates. (B) Maf1 mutant parasites display minimal differences in death rate over the first 72 h of starvation. Regression models were fitted to the 216-h isoleucine starvation data to determine the rate of death of NF54 (WT) and Maf1 mutant (PB-11) parasites over the full 216 h or for only the first 72 h of the same data set (t 1/10 = time required to reach 1/10 the starting level of parasitemia). (C) Maf1 mutant parasites lose the ability to recover after (on average) 43 h of isoleucine starvation. Synchronous young ring-stage PB-11 parasites were washed and transferred to medium lacking isoleucine. Every 3 h, samples were transferred to normal medium for a 72-h recovery period. The final level of parasitemia after recovery was quantified by flow cytometry. A logistic regression fitted to the data shows that each hour of starvation decreases the parasitemia level to 93% of the level seen the previous hour (β o = −0.076, P < 2.00 × 10 −16 ). The logistic model fitted to the data predicts that the time point corresponding to 43 h of starvation is the point at which 50% of the PB-11 parasites are able to recover and 50% are not (t 50 ). Data shown are the results of three biological replicates.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Flow Cytometry

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: PB-11 Maf1 mutant parasites display defects in recovery from fosmidomycin exposure and low-temperature treatment. (A) Young ring-stage NF54 (WT) and Maf1 mutant (PB-11) parasites were incubated in the presence of 5 μM fosmidomycin (fosm.) for 72 h and were then “recovered” by incubation for a further 72 h in the presence of 5 μM fosmidomycin and 5 μM geranylgeraniol. Growth was measured relative to that of parasites incubated for 72 h in normal medium treated with both fosmidomycin and geranylgeraniol. (B) Young ring-stage parasites were incubated at 18°C for 72 h and were recovered by 72 h of growth at 37°C. Growth was measured relative to that of parasites incubated for 72 h at 37°C. P values were calculated using t tests of three biological replicates.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Incubation

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: PB-11 Maf1 parasites display a decreased growth rate under low-isoleucine conditions and at elevated temperatures. Growth curves were measured for NF54 (WT) and Maf1 mutant (PB-11) parasites cultured in normal culture medium at 37°C ( P < 0.0001) (A), 8 μM isoleucine (approximately 2% the concentration of normal medium) ( P < 0.001) (B), normal culture medium at 39°C ( P = 0.003) (C), and 20% (0.4 g/liter) of the glucose level of normal medium (2.0 g/liter) ( P = 0.08) (D). P values represent results of tests of the parasite line (i.e., the wild type versus PB-11) as a factor in the regression analysis. t D , doubling time.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Cell Culture, Concentration Assay

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: Maf1 mutant parasites display elevated pre-tRNA expression and elevated global translation under normal and isoleucine starvation conditions. (A) An alignment of the genomic pre-tRNA Tyr sequence and the mature tRNA Tyr sequence reveals an 11-nucleotide intron adjacent the anticodon. (B) Stem-loop RT-qPCR profiling of pre-tRNA expression in NF54 (WT) and Maf1 mutant (PB-11) parasites. Synchronous young ring-stage parasites were incubated in normal medium or medium lacking isoleucine for 24 h prior to RNA isolation. Pre-tRNA Tyr expression was quantified relative to 5.8S rRNA levels. (C) ELISA of puromycin incorporation relative to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene levels in wild-type and mutant parasites under conditions of the indicated treatments. Synchronous young ring-stage parasites were incubated in normal medium or medium lacking isoleucine for 24 h prior to the 1-h puromycin pulse and subsequent harvesting. Translational arrest by cycloheximide (CHX) treatment was used as a negative control. P values were calculated using t tests of three biological replicates.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Expressing, Sequencing, Quantitative RT-PCR, Incubation, Isolation, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A Phase contrast microscope images showing the phenotype and morphology of the cells in the course of conversion of fibroblasts to iHeps at different times points after transduction with a cocktail of three TFs HNF1A, HNF4A and FOXA3 . B Generation of highly proliferative iHep cells by transducing iHeps with two pools of liver cancer-specific oncogenic drivers, a list of xenograft experiments in nude mice that were used to test the tumorigenicity of different conditions, and mutation rates of the oncogenic drivers as reported in the COSMIC database for HCC and MYC amplification as reported in . CMT pool contains three oncogenes CTNNB1 T41A , MYC, and TERT, and CMT + sg TP53 pool contains the same oncogenes along with constructs for TP53 inactivation by CRISPR-Cas9. Phase contrast microscope images showing the phenotype and morphology of the cells. Oncogenes are co-transduced with fluorescent reporter mCherry for the detection of transduced cells. Oncogene transduction to fibroblasts fails to transform the cells, passaging of oncogene-expressing fibroblasts results in cellular senescence as demonstrated by β-galactosidase staining and loss of mCherry-positive oncogene-expressing cells from the fibroblast population. iHeps maintained in defined culture medium become senescent around week four of transdifferentiation although they can survive in culture for several weeks after that if not passaged. Passaging of iHeps without oncogenes results in apoptosis after few passages. Scale bar 1000 μm unless otherwise specified.

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Microscopy, Transduction, Mutagenesis, Amplification, Construct, CRISPR, Passaging, Expressing, Staining

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A Subcutaneous injection of transformed iHeps results in xenograft tumors in nude mice (tumor size of 1.5 cm ~23 weeks after xenotransplantation). Proliferative iHeps transduced with defined CMT oncogenes with TP53 inactivation (CMT + sg TP53 ) or control iHeps without oncogenes were used in the injections. B In vivo imaging of xenograft tumors ~12 weeks after implantation. Two biological replicate experiments are shown for CMT + sg TP53 cells with iHep conversion and oncogene transduction with TP53 inactivation performed in two separate human fibroblast cell lines (foreskin fibroblast [left panel] and fetal lung fibroblast [middle]) as well as proliferative CMT iHeps without TP53 inactivation (right). Fluorescence signal emitted by mCherry co-transduced with the oncogenes is detected in vivo using the Lago system (scale bar = radiance units). Control mice are injected with either fibroblasts or iHeps. C Histological analysis of CMT + sg TP53 tumor tissue harvested at 20 weeks. Hematoxylin-eosin (H&E) staining for general histology and immunohistochemical staining for Ki-67 for cell proliferation (100x magnification). Note that the appearance of the tumor is consistent with both poorly differentiated hepatic tumor or sarcoma. Differential diagnosis from sarcoma is accomplished by analysis of marker gene expression (see Figs. and ). D Analysis of chromosomal aberrations in the transformed iHeps by spectral karyotyping. CMT + sg TP53 cells were analyzed at passage 18 (early) and passage 50 (late) and CMT cells at passage 18. Fibroblasts have normal diploid karyotype (46, XY, representative spectral image on left) and transformed iHeps show aneuploidies as indicated in the figure. Early passage CMT + sg TP53 cells show two different populations with two distinct modal chromosome numbers (45, XY and 67-92, XY, representative spectral image for 45, XY on middle-left). Late passage CMT + sg TP53 cells have modal chromosome number 67-92, XY (middle-right) and CMT cells 75, XY (right). In the text box below the images, recurrent chromosomal aberrations seen in majority (>90%) of the cells are reported. E Frequencies of chromosomal alterations reported for human HCC samples [see ].

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Injection, Transformation Assay, Transduction, Control, In Vivo Imaging, Fluorescence, In Vivo, Staining, Immunohistochemical staining, Biomarker Discovery, Marker, Gene Expression

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A Gene set enrichment analysis (GSEA) results for CMT-iHeps and CMT + sg TP53 -iHeps compared to control fibroblasts against liver cancer signature [HCC Subclass 2 ] from molecular signatures database (MSigDB). Positive normalized enrichment score (NES) reflects overrepresentation of liver cancer signature genes among the top-ranked differentially expressed genes in CMT-iHep and CMT + sg TP53 -iHep conditions compared to control fibroblasts. B Differential expression levels [log 2 (fold change)] of marker genes for fibroblasts, hepatocytes, and liver cancer in bulk RNA-seq measurements from CMT + sg TP53 -iHeps and CMT-iHeps at p20 (~22 weeks after oncogene transduction) as well as xenograft tumor from CMT + sg TP53 cells against control fibroblasts (mean ± standard error, n = 3). C IGV snapshots for promoter regions of representative genes from fibroblast markers ( MMP3 ), liver markers ( SERPINA1/α-1-antitrypsin ), and liver cancer markers ( SAA1 ) showing ATAC-seq enrichment from fibroblast and CMT + sg TP53 -iHeps. D Chromatin accessibility and CpG methylation of DNA measured using NaNoMe-seq. Cytosine methylation detected using Nanopore sequencing from CMT + sg TP53 -iHeps and control fibroblasts is shown for promoter regions of representative genes from fibroblast markers ( MMP3 ), liver markers ( SERPINA1/α-1-antitrypsin ), and liver cancer markers ( SAA1 ) using a window of TSS ± 1500 bp. GpCpH methylation (all GC sequences where the C is not part of a CG sequence also, top) reports on chromatin accessibility, whereas HpCpG methylation reports on endogenous methylation of cytosines in the CpG context. E CpG methylation detected using bisulfite-sequencing from primary human foreskin fibroblasts and from the normal adult liver [data from the Roadmap Epigenomics Consortium ]. IGV snapshots from the genomic loci corresponding to the MMP3 , SERPINA1 , and SAA1 promoters (same regions as indicated in Fig. 5D) showing methylation proportions [methylated calls/(methylated calls + unmethylated calls)] for all CpGs covered by at least 4 reads.

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Control, Quantitative Proteomics, Marker, RNA Sequencing, Transduction, CpG Methylation Assay, Methylation, Nanopore Sequencing, Sequencing, Methylation Sequencing

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A Immunohistochemical analysis of xenograft tumor tissue from CMT + sg TP53 harvested at 20 weeks and xenograft tumor from the HuH7 HCC cell line. Staining for glypican-3 and arginase-1 are shown along with negative control without primary antibody (100x magnification). B (Top) Beta-galactosidase staining as a marker of cellular senescence in primary human hepatocytes (control), after transduction of CMT oncogenes, or after transduction with iHep-TFs (HNF1A, HNF4A, FOXA3) followed by CMT oncogene transduction one week later (stained three weeks after first transduction). (Middle) Beta-galactosidase staining as a marker of cellular senescence in control fibroblasts and fibroblasts transduced with CMT oncogenes and stained at p4. (Bottom) Fluorescent microscope images of induced neurons with and without transduction of neuroblastoma-specific oncogenes (at three weeks of neuronal differentiation) visualized using EGFP at ten weeks after neuronal conversion. C Schematic presentation of the molecular approach for identifying minimal determinants of tumorigenesis in specific tissues. Lineage-specific transcription factors are used to reprogram human fibroblasts to precise cellular identity (left), whose transformation by specific combinations of oncogenes (right) can then be tested. This approach combined with single-cell RNA-seq and RNA velocity analyses allows also analysis of which cell type along the stem cell to terminally differentiated cell axis (top to bottom) is susceptible for transformation.

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Immunohistochemical staining, Staining, Negative Control, Marker, Control, Transduction, Microscopy, Transformation Assay, RNA Sequencing

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A , B t-SNE plots of 3,500 single cells from fibroblasts, iHeps at one to three weeks after iHep induction, iHeps transduced with CMT oncogenes at one week and harvested for scRNA-seq two weeks later, and fibroblasts transduced with CMT oncogenes and harvested at one and three weeks. Cells are colored by sample ( A ), and distinct clusters ( B ) based on their expression profiles with sample collection time points indicated. C Principal component analysis (PCA) projection of single cells from control fibroblasts, iHeps at one to three weeks after iHep induction, and CMT-iHeps two weeks after oncogenes shown with velocity field with the observed states of the cells shown as circles and the extrapolated future states shown with arrows for the first two principal components. Cells are colored by cluster identities corresponding to Fig. 4B. D Relative expression of the genes from the Notch signaling pathway (panel on the right) across pseudotime in the single-cell RNA-seq data from control fibroblasts, iHeps at one to three weeks after iHep induction, and CMT-iHeps two weeks after oncogenes (the expression of a gene in a particular cell relative to the average expression of that gene across all cells). Relative expression of the senescence marker genes (panel on the left) from control fibroblasts and fibroblasts transduced with CMT oncogenes and harvested at one and three weeks after transduction. Color codes illustrating sample and cluster identities correspond to the colors in Fig. 4A, B, respectively. E Expression levels [log 2 (transcripts per million, tpm)] for LGR5 as well as lentiviral and endogenous HNF4A, TERT , and CTNNB1 in bulk RNA-seq measurements from control fibroblasts, iHeps at four weeks of differentiation, CMT + sg TP53 -iHeps at two and 22 weeks after oncogene transduction, xenograft tumor from CMT + sg TP53 cells, and from liver cancer cell lines HepG2 and HuH7 (mean ± standard error, n = 3). Nanopore sequencing was performed from the CMT + sg TP53 cells at 22 weeks after oncogene transduction as indicated in the figure and used for identifying the genomic insertions of the lentiviral constructs (Supplementary Table ).

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Transduction, Expressing, Control, RNA Sequencing, Marker, Nanopore Sequencing, Construct

Journal: Frontiers in Plant Science

Article Title: Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

doi: 10.3389/fpls.2017.01347

Figure Lengend Snippet: IFR1 localizes to the cytosol in Chlamydomonas reinhardtii cells. Laser scanning confocal microscopy detection of subcellular localization of the mVenus (yellow) fluorescent reporter fused to N- or C-terminus of IFR1 (N/C). A cell line expressing mVenus in the cytosol (Cyto, ) and the parental strain (PCS) served as controls. Individual imaging channels are presented, YFP: mVenus reporter signal in the yellow range, Chlorophyll: autofluorescence of chlorophyll visualized in the red range and used to orient the cells, Overlay: YFP and Chloro channel overlay, DIC: differential interference contrast. Scale bars represent 5 μm.

Article Snippet: To heterologously express IFR1 in Escherichia coli , a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the Nde I and Xho I restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Techniques: Confocal Microscopy, Expressing, Imaging

Journal: Frontiers in Plant Science

Article Title: Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

doi: 10.3389/fpls.2017.01347

Figure Lengend Snippet: IFR1 accumulation is triggered by the SOR1-dependent pathway. (A) Samples were taken before (0 h, +S) and during the course of hydrogen production induced by sulfur deprivation of a wild type cell line (16–48 h; –S). IFR1 accumulation was analyzed with an IFR1-specific antiserum (αIFR1) and equal protein loading confirmed by colloidal Coomassie staining (CBB). (B) Comparison of IFR1 mRNA levels in the sor1 mutant and its parental strain (4A+) with samples taken in the late exponential phase. mRNA levels were determined by RTqPCR and the IFR1 transcript level in 4A+ was set to 1. Median and interquartile range shown in the box-and-whisker diagram are derived from two biological replicates, each including nine technical replicates ( n = 18). (C) Representative immunoblot (αIFR1) showing IFR1 accumulation during growth of mutant sor1 and its parental strain (4A+) in nutrient-replete TAP medium for 3 days. Relative band intensities ( Dens. ) determined by densitometric scanning of immunblot signals are given relative to the band intensity of the 4A+ sample at t 48 h (set to 1). (D) Position of the octanucleotide motif CAACGTTG described to represent an electrophile response element (ERE; ) implicated in the genetic response to reactive electrophile species (RES) and SOR1-dependent signaling relative to the start codon (ATG) of the 4.87 kbp IFR1 gene, comprising exons, introns and untranslated regions (UTRs). (E) IFR1 mRNA levels determined by RTqPCR following dark treatment of WT cell cultures with DBMIB (5 μM) and 2-( E )-hexenal (500 μM) for 24 h. The mRNA level of the solvent control sample was set to 1. Median and interquartile range shown in the box-and-whisker diagram are derived from two biological replicates, each including six technical replicates ( n = 12). (F) Immunoblot (αIFR1) showing IFR1 accumulation distinct time points (6–24 h) after the addition of DBMIB (5 μM) or only solvent (Control) to a liquid TAP culture of the C. reinhardtii wild type CC124 and subsequent dark incubation for 24 h.

Article Snippet: To heterologously express IFR1 in Escherichia coli , a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the Nde I and Xho I restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Techniques: Staining, Comparison, Mutagenesis, Whisker Assay, Derivative Assay, Western Blot, Solvent, Control, Incubation

Journal: Frontiers in Plant Science

Article Title: Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

doi: 10.3389/fpls.2017.01347

Figure Lengend Snippet: IFR1 knock-down causes diminished tolerance toward RES in C. reinhardtii. (A) Immunodetection of IFR1 protein (αIFR1) in the parental strain (PCS; wild type CC124) and IFR1 knock-down strains (IFR1_1 and IFR1_6) detected after 48 h of cultivation in sulfur deplete medium. A colloidal Coomassie stained gel (CBB) served as loading control. Different amounts of proteins were used and band intensities (lower bar diagram) determined by densitometric analysis (1x PCS set to 100%). (B,C) IFR1 accumulation in PCS and IFR1 knock-down strains grown for 24 h in TAP supplemented with DBMIB (5 μM) or 2-(E)-hexenal (500 μM). (D) Growth inhibition by reactive oxygen species determined for the PCS and the two IFR1 knock-down strains during 24 h of growth in TAP supplemented with 4 μM rose Bengal (RB), 15 μM neutral red (NR), or 0.5 μM methyl viologen (MV). Optical densities (determined at 680 and 750 nm) and cell counts are given relative to the untreated/solvent-control sample (set to 1). Error bars indicate standard errors derived from three biological replicates including technical replicates ( n = 3). Asterisks indicate significant differences between PCS and knock-down strains according to a two-tailed Student’s t -test ( p < 0.05). (E,F) Growth inhibition following treatment of PCS and IFR1 knock-down strains with 5 μM DBMIB and 500 μM 2-(E)-hexenal for 9 or 24 h in TAP medium. Standard errors are derived from three biological replicates, including technical replicates ( n = 3). Except for the data indicated by asterisks ( p > 0.05) differences between PCS and knock-down strains were significant according to a two-tailed Student’s t -test ( p < 0.05).

Article Snippet: To heterologously express IFR1 in Escherichia coli , a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the Nde I and Xho I restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Techniques: Knockdown, Immunodetection, Staining, Control, Inhibition, Solvent, Derivative Assay, Two Tailed Test

Journal: Frontiers in Plant Science

Article Title: Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

doi: 10.3389/fpls.2017.01347

Figure Lengend Snippet: Prolonged hydrogen production in IFR1 knock-down strains compared to the wild type. (A) Time course of hydrogen production for the parental strain (PCS) and IFR1 knock-down strains. Hydrogen yields in the knock-down strains are given relative to the final yield of the parental strain (set to 100%). Each data curve represents an average of three biological replicates including three technical triplicates ( n = 9) with error bars representing the standard error. (B) H 2 production rates during the course of hydrogen production. Error bars indicate the standard error ( n = 9) and asterisks indicate differences between PCS and knock-down strains which are significant according to a two-tailed Student’s t -test ( ∗ p < 0.05).

Article Snippet: To heterologously express IFR1 in Escherichia coli , a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the Nde I and Xho I restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Techniques: Knockdown, Two Tailed Test

Journal: Frontiers in Plant Science

Article Title: Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

doi: 10.3389/fpls.2017.01347

Figure Lengend Snippet: Contribution of PSII and photosynthetic/respiration (P/R) rates on hydrogen production. (A) Maximum quantum yield (F v /F m ) of dark-adapted cells of the parental strain (PCS) and IFR1 knock-down strains (IFR1_1/IFR1_6) before (t 0 ) and after exposure to sulfur limitation (t 24 –t 168 h ) and aerobic conditions. Error bars indicate the standard error from three biological replicates ( n = 3). (B) Time course of the maximum quantum yield (F v /F m ; left y-axis) and the cellular chlorophyll content (right y-axis) during photosynthetic hydrogen production of the parental strain (PCS) and one of the IFR1 knock-down strains (IFR1_6). Chlorophyll data were normalized to the chlorophyll content of PCS at t 0 (set to 100%). Standard errors derived from three biological replicates ( n = 3) are indicated as error bars. Except for t 0 , the differences between PCS and IFR1_6 in regard to F v /F m were significant according to a two-tailed Student’s t -test ( p < 0.05). (C) Representative immunoblot showing the immunodetection of PSII subunit D1 (upper left panel; αD1) in samples of the parental strain (PCS) and IFR1_6 taken at indicated times during a hydrogen production experiment. A colloidal Coomassie stain (lower left panel; CBB) served as a loading control. Results from densitometric scanning (right panel) of blot signals are given relative to the D1 signal intensity determined for t 0 (set to 100%). Error bars indicate standard errors (three biological replicates; n = 3). (D) Relative H 2 yields obtained with the parental control strain (PCS) (black bars) and knock-down strain IFR1_6 (gray bars) in the absence or presence of 20 μM DCMU. Hydrogen yields determined for the untreated PCS were set to 100%. Error bars represent standard error ( n = 6).

Article Snippet: To heterologously express IFR1 in Escherichia coli , a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the Nde I and Xho I restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Techniques: Knockdown, Derivative Assay, Two Tailed Test, Western Blot, Immunodetection, Staining, Control

Journal: Frontiers in Plant Science

Article Title: Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

doi: 10.3389/fpls.2017.01347

Figure Lengend Snippet: Knock-down of IFR1 in the boosts hydrogen production in the high hydrogen producer mutant stm6. (A) Immunoblot analysis of IFR1 accumulation in stm6 and stm6_IFR1kd cultivated under sulfur-limiting conditions. Different amounts of total protein (1X; 1.5X; and 2X) were used for immunodetection of IFR1 (αIFR1) with a colloidal Coomassie stain (CBB) serving as a loading control. Results from densitometric signal analysis (dens.) are indicated. (B) Relative time-dependent H 2 yields of the stm6 parental strain (black curve) and stm6_IFR1kd (gray curve) with the final yield in stm6 set to 100%. Error bars represent the standard error (three biological replicates including technical triplicates, n = 9). (C) Maximum quantum yield of PSII determined after dark incubation (Fv/Fm) determined in cultures of stm6 (black bars) and stm6_IFR1kd (gray bars) exposed to sulfur starvation. Standard errors, shown as error bars are derived from three biological replicates including technical duplicates ( n = 6). Except for t 0 , differences between stm6 and stm6_IFR1kd were significant according to a two-tailed Student’s t -test ( p < 0.05).

Article Snippet: To heterologously express IFR1 in Escherichia coli , a codon optimized full length IFR1 coding sequence (phytozome: Transcript Cre11.g477200.t1.2) was synthesized de novo (Genscript, United States) and cloned between the Nde I and Xho I restriction sites of expression vector pET-24a(+) (Novagen), enabling streptag-based affinity purification.

Techniques: Knockdown, Mutagenesis, Western Blot, Immunodetection, Staining, Control, Incubation, Derivative Assay, Two Tailed Test