Additional file 1: Figure S1. of Analysis of archived residual newborn screening blood spots after whole genome amplification

Concordance Rates. Plot represents the concordance rates between technical replicates and biological replicates (wgaDNA vs gDNA), where positions are only considered, when sequencing depth is greater than the Coverage (X-axis). Plus sign and down triangle are additionally filtered by the GATK hard filter

Polyamorphism Mirrors Polymorphism in the Liquid–Liquid Transition of a Molecular Liquid

Liquid–liquid transitions between two amorphous phases in a single-component liquid have courted controversy. All known examples of liquid–liquid transitions in molecular liquids have been observed in the supercooled state, suggesting an intimate connection with vitrification and locally favored structures inhibiting crystallization. However, there is precious little information about the local molecular packing in supercooled liquids, meaning that the order parameter of the transition is still unknown. Here, we investigate the liquid–liquid transition in triphenyl phosphite and show that it is caused by the competition between liquid structures that mirror two crystal polymorphs. The liquid–liquid transition is found to be between a geometrically frustrated liquid and a dynamically frustrated glass. These results indicate a general link between polymorphism and polyamorphism and will lead to a much greater understanding of the physical basis of liquid–liquid transitions and allow the systematic discovery of other examples

SB505124 induces bradyzoite development.

<p>A. HFFs growing in 24 well plates were infected with RH, GT1, Pru, and CTG. After 5–7 days, monolayers were methanol-fixed, stained with crystal violet, and plaques were counted. Means and standard deviations are shown. B. HFFs grown on glass coverslips were infected with Pru strain parasites and either mock-treated or treated with pH 8.2 media or 3 µM SB505124. After 72 h, cells were fixed and stained with anti-SAG1 antisera and Dolichos-FITC to visualize cysts. Shown are representative images. C. HFFs were infected with Pru and either mock-treated or treated with pH 8.2 media or 3 µM SB505124. After 72 h, parasites were released from host cells, washed, and lysed. Total RNA was extracted, DNase I-treated and converted to cDNA. Relative amounts of transcripts were determined by Real Time PCR using β-actin as an internal reference. Fold change for each gene's transcript abundance is reported as 2^−ΔΔCt. Shown are averaged data with standard deviations from 3 experiments. Note that changes in ENO2 are not statistically significant.</p

Forward Genetic Screening Identifies a Small Molecule That Blocks <i>Toxoplasma gondii</i> Growth by Inhibiting Both Host- and Parasite-Encoded Kinases

<div><p>The simultaneous targeting of host and pathogen processes represents an untapped approach for the treatment of intracellular infections. Hypoxia-inducible factor-1 (HIF-1) is a host cell transcription factor that is activated by and required for the growth of the intracellular protozoan parasite <i>Toxoplasma gondii</i> at physiological oxygen levels. Parasite activation of HIF-1 is blocked by inhibiting the family of closely related Activin-Like Kinase (ALK) host cell receptors ALK4, ALK5, and ALK7, which was determined in part by use of an ALK4,5,7 inhibitor named SB505124. Besides inhibiting HIF-1 activation, SB505124 also potently blocks parasite replication under normoxic conditions. To determine whether SB505124 inhibition of parasite growth was exclusively due to inhibition of ALK4,5,7 or because the drug inhibited a second kinase, SB505124-resistant parasites were isolated by chemical mutagenesis. Whole-genome sequencing of these mutants revealed mutations in the <i>Toxoplasma</i> MAP kinase, TgMAPK1. Allelic replacement of mutant TgMAPK1 alleles into wild-type parasites was sufficient to confer SB505124 resistance. SB505124 independently impacts TgMAPK1 and ALK4,5,7 signaling since drug resistant parasites could not activate HIF-1 in the presence of SB505124 or grow in HIF-1 deficient cells. In addition, TgMAPK1 kinase activity is inhibited by SB505124. Finally, mice treated with SB505124 had significantly lower tissue burdens following <i>Toxoplasma</i> infection. These data therefore identify SB505124 as a novel small molecule inhibitor that acts by inhibiting two distinct targets, host HIF-1 and TgMAPK1.</p></div

SB505124 reduces parasite growth in <i>Toxoplasma</i>-infected mice.

<p>RH-GFP infected mice were intraperitoneally injected daily with 10 mg/kg SB505124 or DMSO alone. After 5 days post-infection, mice were sacrificed and flow cytometric analysis was performed on peritoneal exudate cells (3–4 mice per treatment group per experiment, 2 independent experiments). A. FACS plots (upper) and histograms (lower) showing percentages of infected (GFP⁺) cells of two representative mice per treatment group. B. Mean percentages of infected cells between treatment groups with standard deviations. C. Relative MFI of infected (GFP⁺) cells with standard deviations. D. ELISA determination of serum IFNγ levels of mock- and drug-treated, intraperitoneally infected mice 5 days post-infection. Shown are average and standard deviations.</p

Additional file 4 of Novel temporal and spatial patterns of metastatic colonization from breast cancer rapid-autopsy tumor biopsies

Additional file 4: Somatic SNVs with ssID reference

Generation of SBR mutants.

<p>A. Relative plaque formation in HFFs was determined for each parasite strain in the presence of increasing concentrations of SB505124. B–D. Parasite replication was measured by infecting HFFs on glass coverslips in the presence or absence of 3 µM SB505124 and then fixing the cells 24 hours later. Parasites and nuclei were detected with anti-SAG1 antibody and DAPI, respectively. B. Representative images. C. For each replicate, 100 vacuoles were monitored for parasites per vacuole and nuclei per parasite. Vacuoles were designated as being irregular if they contained an irregular number of parasites/vacuole (non 2ⁿ). Shown are averaged percentages and standard deviations of 2 independent experiments with two replicates each. D. Averaged percentages and standard deviations of irregular vacuoles (show in C) by nuclei per parasite.</p

Additional file 3 of Novel temporal and spatial patterns of metastatic colonization from breast cancer rapid-autopsy tumor biopsies

Additional file 3: Subclone analysis-alternative solutions

TgMAPK1 is an SBR gene.

<p>A. Venn diagram of whole genome sequencing data of codon-changing SNVs identified in each mutant. B. Amino acid positions of TgMAPK1^SBR mutations. C. TgMAPK1^SBR allelic replacement strategy. Primers 1 and 2 were used to amplify 944 bp fragments of genomic DNA containing the SBR allele and cloned into pCR2.1. Primers 3 and 4 were used to amplify 1055 bp fragments of genomic DNA to confirm allelic replacement by Sanger sequencing. D. RHΔku80 parasites were transfected with linearized TgMAPK1^WT or TgMAPK1^SBR replacement constructs and grown in 3 µM SB505124-treated HFFs. Shown are representative images depicting the ability of RHΔKu80:TgMAPK1^SBR1 to grow and form plaques after 5 days of growth in the presence of 3 µM SB505124.</p