Identifying coronary artery progenitors and their developmental pathways could inspire novel regenerative treatments for heart disease. Multiple sources of coronary vessels have been proposed, including the sinus venosus (SV), endocardium and proepicardium, but their relative contributions to the coronary circulation and the molecular mechanisms regulating their development are poorly understood. We created an ApjCreER mouse line as a lineagetracing tool to map SV-derived vessels onto the heart and compared the resulting lineage pattern with endocardial and proepicardial contributions to the coronary circulation. The data showed a striking compartmentalization to coronary development. ApjCreER-traced vessels contributed to a large number of arteries, capillaries and veins on the dorsal and lateral sides of the heart. By contrast, untraced vessels predominated in the midline of the ventral aspect and ventricular septum, which are vessel populations primarily derived from the endocardium. The proepicardium gave rise to a smaller fraction of vessels spaced relatively uniformly throughout the ventricular walls. Dorsal (SV-derived) and ventral (endocardialderived) coronary vessels developed in response to different growth signals. The absence of VEGFC, which is expressed in the epicardium, dramatically inhibited dorsal and lateral coronary growth but left vessels on the ventral side unaffected. We propose that complementary SV-derived and endocardial-derived migratory routes unite to form the coronary vasculature and that the former requires VEGFC, revealing its role as a tissue-specific mediator of blood endothelial development.

Most vertebrate organs are composed of epithelium surrounded by support and stromal tissues formed from mesenchyme cells, which are not generally thought to form organized progenitor pools. Here we use clonal cell labeling with multicolor reporters to characterize individual mesenchymal progenitors in the developing mouse lung. We observe a diversity of mesenchymal progenitor populations with different locations, movements, and lineage boundaries. Airway smooth muscle (ASM) progenitors map exclusively to mesenchyme ahead of budding airways. Progenitors recruited from these tip pools differentiate into ASM around airway stalks; flanking stalk mesenchyme can be induced to form an ASM niche by a lateral bud or by an airway tip plus focal Wnt signal. Thus, mesenchymal progenitors can be organized into localized and carefully controlled domains that rival epithelial progenitor niches in regulatory sophistication.

We report the site-specific incorporation of a thiocyanate vibrational probe into the active site oxyanion hole of ketosteroid isomerase (KSI) to test the effect of hydrophobic steroid binding and solvent exclusion on the local electrostatic environment at this position. While binding of an uncharged ground state steroid analog shifts the observed -CN vibrational frequency by +0.4 cm −1 relative to unliganded KSI, binding of an intermediate steroid analog containing localized negative charge results in a +2.8 cm −1 shift. Based on a Stark tuning rate of 0.7 cm −1 /(MV/cm), this shift indicates a fivefold larger change in the projection of the local electric field along the -CN bond in the presence of the charged ligand. Binding of a single ring phenolate with oxyanion charge localization equivalent to the intermediate steroid analog but lacking distal hydrocarbon rings results in an identical -CN peak shift. We conclude that solvent exclusion and replacement by hydrophobic steroid rings negligibly alter the electrostatic environment within the KSI oxyanion hole. Development of localized negative charge analogous to that of the dienolate intermediate during steroid isomerization dramatically increases the magnitude of the local electric field. This increase reflects field contributions from the localized negative charge itself as well as possible increased ordering of active site dipoles in response to charge localization.The thousands of enzyme structures solved to date have consistently revealed that biological catalysis occurs within sequestered pockets containing complex interdigitations of polar and hydrophobic groups and from which water molecules are displaced upon substrate binding. 1 This chemical complexity has sparked considerable controversy regarding the electrostatic nature of active sites and the role of substrate binding and solvent exclusion in shaping active site electrostatics. [2][3][4][5][6][7] We report herein the site-specific incorporation of a thiocyanate vibrational probe 8 into the active site of Pseudomonas putida ketosteroid isomerase (KSI) to directly and quantitatively test the effect of steroid binding and concomitant solvent exclusion on the local electrostatic environment.KSI catalyzes a double-bond migration reaction in steroid substrates that involves formation of a dienolate intermediate within an active site oxyanion hole composed of Y16, protonated D103, and a preponderance of hydrophobic residues (Scheme 1A and Figure S1). Numerous physical changes occur upon steroid binding that might alter the local electrostatic environment within the KSI active site. In the free enzyme an ordered water molecule is sboxer@stanford.edu, herschla@stanford.edu. Supporting Information Available: Experimental methods and Figures S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2011 September 12. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Auth...

Objective: To develop a microarray-based method for noninvasive prenatal testing (NIPT) and compare it with next-generation sequencing. Methods: Maternal plasma from 878 pregnant women, including 187 trisomy cases (18 trisomy 13, 37 trisomy 18, 132 trisomy 21), was evaluated for trisomy risk. Targeted chromosomes were analyzed using Digital Analysis of Selected Regions (DANSR™) assays. DANSR products were subsequently divided between two DNA quantification methods: microarrays and next-generation sequencing. For both microarray and sequencing methodologies, the Fetal-Fraction Optimized Risk of Trisomy Evaluation (FORTE™) algorithm was used to determine trisomy risk, assay variability across samples, and compute fetal fraction variability within samples. Results: NIPT using microarrays provided faster and more accurate cell-free DNA (cfDNA) measurements than sequencing. The assay variability, a measure of variance of chromosomal cfDNA counts, was lower for microarrays than for sequencing, 0.051 versus 0.099 (p < 0.0001). Analysis time using microarrays was faster, 7.5 versus 56 h for sequencing. Additionally, fetal fraction precision was improved 1.6-fold by assaying more polymorphic sites with microarrays (p < 0.0001). Microarrays correctly classified all trisomy and nontrisomy cases. Conclusions: NIPT using microarrays delivers more accurate cfDNA analysis than next-generation sequencing and can be performed in less time.

Objective: To determine the performance of a targeted microarray-based cell-free DNA (cfDNA) test (Harmony Prenatal Test®) for the identification of pregnancies at increased risk for 22q11.2 deletion. Methods: Test performance was determined in 2 steps including a total of 1,953 plasma samples. Analytical validation was performed in 1,736 plasma samples. Clinical verification of performance was performed in an additional 217 prospectively ascertained samples from pregnancies with fetal deletion status determined by diagnostic testing. Results: Analytical sensitivity was 75.4% (95% CI: 67.1–82.2%) based on 122 samples with deletions ranging from 1.96 to 3.25 Mb. In 1,614 presumed unaffected samples, specificity was determined to be at least 99.5% (95% CI: 99.0–99.7%). In the clinical cohort, 5 of 7 samples from pregnancies affected with 22q11.2 deletion were determined to have a high probability of deletion. There were no false positive results in the 210 unaffected samples in this cohort. These clinical data are consistent with the performance demonstrated in the analytical validation. Conclusions: cfDNA testing using a targeted microarray-based technology is able to identify pregnancies at increased risk for 22q11.2 deletions of 3.0 Mb and smaller while maintaining a low false positive rate.

Objective To assess the performance of a directed chromosomal analysis approach in the prenatal evaluation of fetal sex chromosome aneuploidy.Methods We analyzed 432 frozen maternal plasma samples obtained from patients prior to undergoing fetal diagnostic testing. The cohort included women greater than 18 years of age with a singleton pregnancy of greater than 10 weeks gestation. Samples were analyzed using a chromosome-selective approach (DANSR TM ) and a risk algorithm that incorporates fetal fraction (FORTE TM ).Results The cohort included 34 cases of sex chromosome aneuploidy. The assay correctly identified 26 of 27 (92.6%) cases of Monosomy X, one case of XXX, and all six cases of XXY. There were four false positive cases of sex chromosome aneuploidy among 380 euploid cases for an overall false positive rate of less than 1%.Discussion Analysis of the risk for sex chromosome aneuploidies can be accomplished with a targeted assay with high sensitivity.

ObjectivesVarious methods of fetal‐fraction measurement have been employed in conjunction with different approaches to cell‐free DNA testing for fetal aneuploidy. In this study, we determined the accuracy and reproducibility of fetal‐fraction measurement using polymorphic assays that are incorporated into the test design as part of the Harmony® prenatal test and evaluated whether the single nucleotide polymorphisms selected for and used in these assays can be applied broadly to all patient populations.MethodsClinical maternal plasma samples were assayed using a custom microarray with Digital ANalysis of Selected Regions (DANSR) assays designed to cover non‐polymorphic targets on chromosomes of interest for aneuploidy assessment (13, 18, 21, X and Y) and polymorphic targets for fetal‐fraction assessment. In a consecutive series of 47 512 maternal plasma samples, fetal‐fraction measurements based on polymorphic assays were compared with those from Y‐sequence quantitation. Reproducibility was examined between first‐ and second‐tube measurements for the same patient sample in 734 cases. The fraction of informative loci was calculated for 13 988 samples.ResultsThere was a strong correlation between fetal fractions determined using the polymorphic assays and using Y‐chromosome sequence quantitation (r = 0.97). Fetal‐fraction measurement between the first and second tubes was highly reproducible (r = 0.98). The fraction of informative loci observed in a clinical series was consistent with predictions based on assay design.ConclusionsThe method based on relative quantitation at polymorphic loci on a microarray is accurate and reproducible for fetal‐fraction estimation and is equally informative across global populations. This study provides a useful benchmark for ensuring the reliability and accuracy of fetal‐fraction measurement. © 2018 Roche Sequencing Solutions. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

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