Journal:

Article Title: Molecular Characterization of the Fragilysin Pathogenicity Islet of Enterotoxigenic Bacteroides fragilis

doi:

Figure Lengend Snippet: Nucleotide sequences used to define the ends of the fragilysin pathogenicity islet. The nontoxigenic sequence was from B. fragilis VPI 2553. Toxigenic sequences across the left and right ends of the pathogenicity islet were from enterotoxigenic B. fragilis strain VPI 13784. Lowercase letters represent where the sequence from the nontoxigenic strain varied from the sequence flanking the ends of the islet. A 17-bp sequence present only in DNA from the nontoxigenic strain is boxed. Arrows indicate the ends of the islet and corresponding nucleotide number. Direct repeats (DR) are underlined.

Article Snippet: Sequencing was performed by fluorescent automated DNA sequence analysis at Commonwealth Biotechnologies, Richmond, Va., and the University of Iowa DNA facility, Iowa City.

Techniques: Sequencing

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: (a) A Clock and Wavefront model: antagonistic gradient of Fgf8 (originating from the posterior PSM, green) and RA (originating from the somites, violet) define a wavefront which interacts with a particular phase of the segmentation clock (in the PSM, red) to generate somites at periodic times and positions. (b) Kymograph of somitogenesis from 7 to 20 somites. The tail elongates at a constant rate V tail while the PSM shrinks at a roughly constant rate V PSM resulting in a somite wavefront propagating at a rate V front = V tail -V PSM .

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques:

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: Molecular models of the determination wavefront. (a) The Goldbeter, Gonze and Pourquié (G 2 P) model assumes that RA directly affects the translation of Fgf8 mRNA into protein, while Fgf8 represses RA via the activation of its degradation enzyme Cyp26. (b) The modified version of the G 2 P model (mG 2 P) proposed here takes into account the observed positive feedbacks of RA on Fgf8 and of Fgf8 on RaldH and the mutual inhibition of RA on MapK (via the RA-mediated activation of Mkp3) and of MapK on RA (via the MapK-controlled activation of Cyp26). (c) Both models predict a bistability of MapK activity for a certain positional range (in grey).

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques: Activation Assay, Modification, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: Time of appearance of somites (t=0 at 7 somites stage). Notice the linearity of the plot, i.e. the regularity of the period of somitogenesis, in all the conditions studied here: WT (n=8), DEAB (an inhibitor of RaldH; with (n=14) or without (n=12) external RA), morpholinos against Fgf8 (MO-Fgf8; n=16), BCI (an inhibitor of Mkp3; n=16) or activation of exogenous Fgf8 (n=17).

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: (a) Staining of phosphorylated MapK by antibodies against this active form in WT embryos at 10 and 15 somites. Notice the smaller domain of activity at 15s as compared to 10s. (b) The data in was quantified by measuring the fluorescence intensity in a single embryo along the antero-posterior axis and averaged over n=17 (10s) and n=21 (15s) embryos. The averaged data is compared to simulations of the mG 2 P model with the parameters of assuming an exponential decrease (see ) between 7s and 10s (or 15s) of the mRNA Fgf8. The x and y-scales were chosen to fit the data at 10s. The simulation results are in qualitative agreement with the data, even though the latter might not be a perfect reflection of the MapK activity level (which depends on the efficiencies of staining and washing).

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques: Staining, Activity Assay, Fluorescence

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: (a) Variation with time of Fgf8 concentration versus somite stage and fit to an exponential decay past 5 somites stage. (b) PSM shortening from 7 somites stage (dots and error bars on mean; n=8) and results (continuous line) of a simulation of the mG 2 P model (with the displayed parameters and assuming an exponential decay of Fgf8 with the timescale measured in (a)). Details in Supp.Mat.

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: Rates of PSM shrinkage (V PSM ), tail growth (V tail ) and wavefront velocity (V front ) in embryos growing (a) without or with morpholinos against Fgf8 (MO-Fgf8; dots and error bars on mean; n=16) injected at one-cell stage or (b) in which an exogenous source of Fgf8 was turned on (n=17). While V PSM is unaffected by MO-Fgf8 and slightly decreases upon over-expression of Fgf8 (in the strong phenotype 2 embryos), V tail decreases in both conditions, resulting in an overall decrease of V front . Continuous lines: simulations of the mG 2 P model with a 60% decrease in Fgf8 mRNA (due to interference with MO-Fgf8; γ=0.8 instead of γ=2 in model, see Supp.Mat.) qualitatively reproduce the data (continuous line in (a)). Similarly simulations with an increasing Fgf8 mRNA of about 2% of the measured increase (see Fig.S7) seem to reproduce the data observed in the strong phenotype 2 case (see Supp.Mat. Fig.S5).

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques: Injection, Over Expression

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: (a,c) MapK activity at steady-state from a simulation of mG 2 P model with the parameters shown in the figures and linear gradients of RaldH (parameter α) and Fgf8 mRNA (parameter γ). Notice the existence of a bistability window (between distances: −40 and −32 in (c)). (b,d) Variation with time of the distance from the tail end (at 0) of the rightmost bistability boundary (red vertical line in (a,c)): assuming linear decrease with time of Fgf8 mRNA (red curve; t ∼ 1 - mF0(t)/mF(0)) or exponential decrease with time of Fgf8 mRNA (blue curve, mF0(t) = mF0(0) e -t ). (e,f) Results of simulations with linear gradients of Cyp26 (parameter β) and Fgf8 mRNA (parameter γ). Notice that the MapK activity levels off at the tail end though a bistability window is still present. (g,h) Results of simulations with a linear gradient of Fgf8 mRNA (parameter γ) only. Notice the increasing MapK activity level and the increased PSM shrinkage rate at the tail end (even assuming an exponential decay with time of FgF8 mRNA).

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: OTTER, a new method quantifying absolute amounts of tRNAs

doi: 10.1101/2020.05.18.101501

Figure Lengend Snippet: ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with dATP and fluorescence-labeled dUTP (such as TMR-dUTP; marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).

Article Snippet: After adding final concentration of 10 μM TMR-dUTP (Roche Diagnostics, Basel, Switzerland), 250 μM dATP, 10 mM MgCl 2 , and 2.0 units of Klenow Fragment (3’-5’ exo − )(New England Biolabs, Ipswich, Massachusetts, USA), total 10 μl of the mixture was incubate at 37°C for 90 min.

Techniques: Fluorescence, Labeling, Activity Assay, Northern Blot