Journal: Genetics

Article Title: Host Mitochondrial Association Evolved in the Human Parasite Toxoplasma gondii via Neofunctionalization of a Gene Duplicate

doi: 10.1534/genetics.115.186270

Figure Lengend Snippet: The T. gondii and H. hammondi MAF1 loci harbor two distinct isoforms while only one isoform is present in N. caninum . (A) Schematic representation of the predicted MAF1 protein. The signal peptide (SP) was predicted using SignalP v4.0 and the putative transmembrane domain (TM) was predicted by TMHMM v2.0. The proline-rich region (Pro-Rich) stretches from AA152 to 164 of TgMAF1RHb1 and is not found within all MAF1 paralogs ( e.g. , TgMAF1RHa1, a2). (B) Phylogram of either cloned MAF1 amino acid sequences from T. gondii , H. hammondi , and N. caninum , or those downloaded directly from ToxoDB (with TG Gene nos.). Cloned sequences of all of the “b” paralogs from T. gondii did not match any predicted gene models in ToxoDB in terms of predicted coding region length and were left out of the analysis. Paralog family is indicated at the end of each name ( e.g. , a1, b1, b2, etc.) (C) d N / d S ratio calculations for all T. gondii “b” MAF1 paralogs, including b0. * indicates significant evidence for diversifying selection for that particular paralog comparison ( P < 0.05).

Article Snippet: A construct encoding the predicted C-terminal domain of TgMAF1RHb1 (Thr159 to Asp435) was codon optimized for Escherichia coli and synthesized by GenScript.

Techniques: Clone Assay, Selection, Comparison

Journal: Genetics

Article Title: Host Mitochondrial Association Evolved in the Human Parasite Toxoplasma gondii via Neofunctionalization of a Gene Duplicate

doi: 10.1534/genetics.115.186270

Figure Lengend Snippet: T. gondii MAF1 paralog expression differs between lineages. (A and B) Polyclonal antibodies were generated specifically against the C termini of TgMAF1RHa1 (Ser173 to Ser443) or TgMAF1RHb1 (Thr159 to Asp435). Protein expression was compared by immunofluorescence across three strains representing clonotypes I, II, and III. Based on immunofluorescence and Western blotting, antibodies against TgMAF1RHa1 detected protein in all three strains, while antibodies against TgMAF1RHb1 detected protein only in RH and CTG (and not ME49). (C) Antibodies against TgMAF1RHb1 are able to detect TgMAF1RHb1 expression in transgenic type II parasites expressing the TgMAF1RHb1 protein.

Article Snippet: A construct encoding the predicted C-terminal domain of TgMAF1RHb1 (Thr159 to Asp435) was codon optimized for Escherichia coli and synthesized by GenScript.

Techniques: Expressing, Generated, Immunofluorescence, Western Blot, Transgenic Assay

Journal: Genetics

Article Title: Host Mitochondrial Association Evolved in the Human Parasite Toxoplasma gondii via Neofunctionalization of a Gene Duplicate

doi: 10.1534/genetics.115.186270

Figure Lengend Snippet: MAF1RHa1 and MAF1RHb1 differ in their ability to complement HMA in T. gondii and N. caninum . (A) HFFs were labeled with MitoTracker and infected with parasites transiently transfected with either HA-MAF1RHa1 or HA-MAF1RHb1. MAF1RHb1 but not MAF1RHa1 is able to confer the HMA phenotype in TgME49. (B) Identical results were obtained for N. caninum . Bar, 5.0 μm. (C) HA-MAF1RHb1 was transfected into either TgME49 (top) or N. caninum (bottom), and HA-positive clones were isolated by limiting dilution. Wild type (WT, left) and TgMAF1RHb1 complemented (right) were grown for 18 hr in HFFs and processed for electron microscopy. Asterisks indicate host mitochondria. Bar, 500 nm.

Article Snippet: A construct encoding the predicted C-terminal domain of TgMAF1RHb1 (Thr159 to Asp435) was codon optimized for Escherichia coli and synthesized by GenScript.

Techniques: Labeling, Infection, Transfection, Clone Assay, Isolation, Electron Microscopy

Journal: Genetics

Article Title: Host Mitochondrial Association Evolved in the Human Parasite Toxoplasma gondii via Neofunctionalization of a Gene Duplicate

doi: 10.1534/genetics.115.186270

Figure Lengend Snippet: N-terminally HA-tagged MAF1 isoforms were expressed in TgME49 parasites and HMA was assessed using MitoTracker or immunofluorescence assay using antibodies against the mitochondrial marker MTCO2. TgMAF1RHb0 and HhMAF1a1 (A and C) did not mediate HMA, while TgMAF1RHb1 and HhMAF1b1 (B and D) are both able to mediate HMA. (E) Quantification of percent vacuole coverage, determined by confocal microscopy. Twenty vacuoles were quantified for each of the MAF1 paralogs indicated, as well as wild-type TgME49. χ 2 P -values: *0.0144; **0.0005; ***<0.0001.

Article Snippet: A construct encoding the predicted C-terminal domain of TgMAF1RHb1 (Thr159 to Asp435) was codon optimized for Escherichia coli and synthesized by GenScript.

Techniques: Immunofluorescence, Marker, Confocal Microscopy

Journal: Genetics

Article Title: Host Mitochondrial Association Evolved in the Human Parasite Toxoplasma gondii via Neofunctionalization of a Gene Duplicate

doi: 10.1534/genetics.115.186270

Figure Lengend Snippet: Expression of TgMAF1RHb1, but not TgMAF1RHa1, in type II T. gondii increases competitive advantage. (A) Mice were infected with mixed populations of TgME49:EV and TgME49:MAF1 with the indicated isoforms and ratios. Infection was allowed to progress for 5 days and population proportions before and after infection were quantified by IFA. Both HMA + and HMA − MAF1 isoforms were assessed. TgME49:TgMAF1RHb1 significantly increases in proportion to TgME49:EV. *χ 2 P -value <0.05. The proportion of TgMAF1RHa1-expressing parasites did not increase during the infection. (B) Percent change per day was calculated for the populations that started with 4:1 TgME49:EV to TgME49:TgMAF1RHb1 both in vitro and in vivo by dividing the total percent increase of TgMAF1RHb expressing parasites within the population by the number of days of infection. The first bar of both the in vitro and in vivo infections represent one clone set, while the second bar for each represents a second clone set. (C) Representative images of a mixed population from A before and after a 5-day in vivo infection. HA staining indicates TgMAF1RHb1-positive vacuoles.

Article Snippet: A construct encoding the predicted C-terminal domain of TgMAF1RHb1 (Thr159 to Asp435) was codon optimized for Escherichia coli and synthesized by GenScript.

Techniques: Expressing, Infection, In Vitro, In Vivo, Staining

Journal: Lipids in Health and Disease

Article Title: In vitro and in vivo plasmalogen replacement evaluations in rhizomelic chrondrodysplasia punctata and Pelizaeus-Merzbacher disease using PPI-1011, an ether lipid plasmalogen precursor

doi: 10.1186/1476-511X-10-182

Figure Lengend Snippet: Ethanolamine plasmalogen levels in RCDP1 and RCDP2 lymphocytes with 0, 20 or 100 μM PPI-1011 for 72 hr . N = 8. Mean ± SEM. All basal decrements and PPI-1011-dependent increases were significantly (p < 0.01) from control lymphocytes and from 0 μM PPI-1011, respectively. 16:0 (palmitic acid), 18:1 (oleic acid), 18:2 (linoleic acid), 20:4 (arachidonic acid), 22:6 (docosahexaenoic acid; DHA).

Article Snippet: Control lymphocytes (Coriell GM13072 and GM02184); PMD lymphocytes (Coriell GM09545; PLP1 c.767C > T or p.P215S); RCDP1 lymphocytes (Coriell GM09291; PEX7 c.870 871insCAA/875T > A or p.C290 E291insQ/L292X and show a severe plasmalogen synthesis defect) and RCDP2 lymphocytes (Coriell GM16776; homozygous for GNPT c.1280-3T > G predicted to encode an in-frame protein p.427 507 del and showing a milder plasmalogen synthesis defect) [ ] were kept as suspension cultures (25 ml flasks) in RPMI 1640 (Hyclone) supplemented with 10% FBS and 1% antibiotic/antimycotic, at 37°C in a 5% CO 2 incubator [ ].

Techniques:

Journal: Lipids in Health and Disease

Article Title: In vitro and in vivo plasmalogen replacement evaluations in rhizomelic chrondrodysplasia punctata and Pelizaeus-Merzbacher disease using PPI-1011, an ether lipid plasmalogen precursor

doi: 10.1186/1476-511X-10-182

Figure Lengend Snippet: Incorporation of PPI-1038 (100 μM; 72 hr) into the 16:0/22:6, 18:0/22:6 and 18:1/22:6 plasmalogens and DHA of RCDP1 and RCDP2 lymphocytes . N = 6. Mean ± SEM. P =[ 13 C 16 ]palmitic acid; G =[ 13 C 3 ]glycerol; D = [ 13 C 3 ]DHA. *, p < 0.01 vs. control.

Article Snippet: Control lymphocytes (Coriell GM13072 and GM02184); PMD lymphocytes (Coriell GM09545; PLP1 c.767C > T or p.P215S); RCDP1 lymphocytes (Coriell GM09291; PEX7 c.870 871insCAA/875T > A or p.C290 E291insQ/L292X and show a severe plasmalogen synthesis defect) and RCDP2 lymphocytes (Coriell GM16776; homozygous for GNPT c.1280-3T > G predicted to encode an in-frame protein p.427 507 del and showing a milder plasmalogen synthesis defect) [ ] were kept as suspension cultures (25 ml flasks) in RPMI 1640 (Hyclone) supplemented with 10% FBS and 1% antibiotic/antimycotic, at 37°C in a 5% CO 2 incubator [ ].

Techniques: