Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment.

During T cell development, multipotent progenitors relinquish competence for other fates and commit to the T cell lineage by turning on Bcl11b, which encodes a transcription factor. To clarify lineage commitment mechanisms, we followed developing T cells at the single-cell level using Bcl11b knock-in fluorescent reporter mice. Notch signaling and Notch-activated transcription factors collaborate to activate Bcl11b expression irrespectively of Notch-dependent proliferation. These inputs work via three distinct, asynchronous mechanisms: an early locus 'poising' function dependent on TCF-1 and GATA-3, a stochastic-permissivity function dependent on Notch signaling, and a separate amplitude-control function dependent on Runx1, a factor already present in multipotent progenitors. Despite their necessity for Bcl11b expression, these inputs act in a stage-specific manner, providing a multitiered mechanism for developmental gene regulation.

Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU.1.

The ETS family transcription factor PU.1 is essential for the development of several blood lineages, including T cells, but its function in intrathymic T-cell precursors has been poorly defined. In the thymus, high PU.1 expression persists through multiple cell divisions in early stages but then falls sharply during T-cell lineage commitment. PU.1 silencing is critical for T-cell commitment, but it has remained unknown how PU.1 activities could contribute positively to T-cell development. Here we employed conditional knockout and modified antagonist PU.1 constructs to perturb PU.1 function stage-specifically in early T cells. We show that PU.1 is needed for full proliferation, restricting access to some non-T fates, and controlling the timing of T-cell developmental progression such that removal or antagonism of endogenous PU.1 allows precocious access to T-cell differentiation. Dominant-negative effects reveal that this repression by PU.1 is mediated indirectly. Genome-wide transcriptome analysis identifies novel targets of PU.1 positive and negative regulation affecting progenitor cell signaling and cell biology and indicating distinct regulatory effects on different subsets of progenitor cell transcription factors. Thus, in addition to supporting early T-cell proliferation, PU.1 regulates the timing of activation of the core T-lineage developmental program.

Cervical tooth anatomy considerations for prefabricated anatomic healing abutment design: A mathematical formulation.

STATEMENT OF PROBLEM: A custom emergence profile offers the ideal horizontal dimensions for an anatomic healing abutment. However, developing such an emergence profile can be a time-consuming and complex process. PURPOSE: The purpose of this study was to develop a mathematical formula defining horizontal cervical tooth geometry to design prefabricated, tooth-specific, healing abutments. MATERIAL AND METHODS: Cone beam computed tomography (CBCT) horizontal cross sections of 989 teeth on 54 participants were measured. For anterior and premolar teeth, 2 perpendicular ellipses were fitted onto the cervical tooth cross section that was defined by 3 parameters. The lingual ellipse followed the lingual outline of the tooth, and its diameter was the largest mesiodistal diameter of the tooth (parameter "a"); its buccolingual radius became parameter "b." The buccal ellipse was perpendicular to the lingual ellipse and followed the buccal outline of the tooth. The buccolingual radius of the smaller ellipse became parameter "c." For molars, the first ellipses followed the mesial outline of the tooth, and its larger diameter (parameter "a") matched the largest buccolingual diameter of the tooth. Its smaller radius became parameter "h1." The second ellipse was parallel to the first ellipse and followed the distal outline of the tooth. Its larger diameter became parameter "b", and its mesiodistal diameter became parameter "h2". Statistical differences between parameters were evaluated by the linear mixed model (α=.05 after Bonferroni adjustment). Pairwise comparisons were made separately for each parameter of the molars and separately for each parameter for the anterior teeth plus premolars. Teeth were put into the same parameter cluster if no significant differences were found between them for a specific parameter. If neither parameter (4 for molars and 3 for the other teeth) was different for 2 teeth, they were put into the same abutment cluster. The abutment clusters determined the type of anatomic healing abutment. The areas were calculated from the developed mathematical formula by using the parameters. In addition, cervical areas of 106 randomly chosen teeth were measured directly with a photo-editing software program. A computer algorithm was used to select 5 CBCT scans from the 54 by using the simple randomization method. The agreement between the 2 methods was evaluated by Bland-Altman analysis. RESULTS: The lower and upper limits of agreement between the 2 methods were -8.57 and 7.36 mm2, respectively, with no bias (-0.61 mm2, P=.224). Significant differences were found between most parameters among the 14 tooth types (P<.001). Based on the parameters, 12 specifically distinct clusters were defined. Two tooth types were pooled into 1 abutment cluster: the maxillary first and second premolars and the mandibular first and second molars. CONCLUSIONS: The cervical tooth cross section can be accurately defined by combining 2 elliptical elements. A comprehensive array of tooth specific emergence profiles can be provided by just 12 different prefabricated abutments, designed as per the recommended parameters.

Whole exome and whole genome sequencing with dried blood spot DNA without whole genome amplification.

Newborn screening (NBS) for rare conditions is performed in all 50 states in the USA. We have partnered with the California Department of Public Health Genetic Disease Laboratory to determine whether sufficient DNA can be extracted from archived dried blood spots (DBS) for next-generation sequencing in the hopes that next-generation sequencing can play a role in NBS. We optimized the DNA extraction and sequencing library preparation protocols for residual infant DBS archived over 20 years ago and successfully obtained acceptable whole exome and whole genome sequencing data. This sequencing study using DBS DNA without whole genome amplification prior to sequencing library preparation provides evidence that properly stored residual newborn DBS are a satisfactory source of DNA for genetic studies.

EXTL3 mutations cause skeletal dysplasia, immune deficiency, and developmental delay.

We studied three patients with severe skeletal dysplasia, T cell immunodeficiency, and developmental delay. Whole-exome sequencing revealed homozygous missense mutations affecting exostosin-like 3 (EXTL3), a glycosyltransferase involved in heparan sulfate (HS) biosynthesis. Patient-derived fibroblasts showed abnormal HS composition and altered fibroblast growth factor 2 signaling, which was rescued by overexpression of wild-type EXTL3 cDNA. Interleukin-2-mediated STAT5 phosphorylation in patients' lymphocytes was markedly reduced. Interbreeding of the extl3-mutant zebrafish (box) with Tg(rag2:green fluorescent protein) transgenic zebrafish revealed defective thymopoiesis, which was rescued by injection of wild-type human EXTL3 RNA. Targeted differentiation of patient-derived induced pluripotent stem cells showed a reduced expansion of lymphohematopoietic progenitor cells and defects of thymic epithelial progenitor cell differentiation. These data identify EXTL3 mutations as a novel cause of severe immune deficiency with skeletal dysplasia and developmental delay and underline a crucial role of HS in thymopoiesis and skeletal and brain development.

Irreversibility of T-Cell Specification: Insights from Computational Modelling of a Minimal Network Architecture.

BACKGROUND/OBJECTIVES: A cascade of gene activations under the control of Notch signalling is required during T-cell specification, when T-cell precursors gradually lose the potential to undertake other fates and become fully committed to the T-cell lineage. We elucidate how the gene/protein dynamics for a core transcriptional module governs this important process by computational means. METHODS: We first assembled existing knowledge about transcription factors known to be important for T-cell specification to form a minimal core module consisting of TCF-1, GATA-3, BCL11B, and PU.1 aiming at dynamical modeling. Model architecture was based on published experimental measurements of the effects on each factor when each of the others is perturbed. While several studies provided gene expression measurements at different stages of T-cell development, pure time series are not available, thus precluding a straightforward study of the dynamical interactions among these genes. We therefore translate stage dependent data into time series. A feed-forward motif with multiple positive feed-backs can account for the observed delay between BCL11B versus TCF-1 and GATA-3 activation by Notch signalling. With a novel computational approach, all 32 possible interactions among Notch signalling, TCF-1, and GATA-3 are explored by translating combinatorial logic expressions into differential equations for BCL11B production rate. RESULTS: Our analysis reveals that only 3 of 32 possible configurations, where GATA-3 works as a dimer, are able to explain not only the time delay, but very importantly, also give rise to irreversibility. The winning models explain the data within the 95% confidence region and are consistent with regard to decay rates. CONCLUSIONS: This first generation model for early T-cell specification has relatively few players. Yet it explains the gradual transition into a committed state with no return. Encoding logics in a rate equation setting allows determination of binding properties beyond what is possible in a Boolean network.

Warming up to composites.

Patient demand for composite restorations has increased dramatically over the past decade. Anterior composites, for teeth that are visible when smiling, have been the standards of practice for many years. For the less visible teeth, it is estimated that the 50% mark for composite restorations in the posterior region was surpassed in 2000.