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In the original version of Figure 5A, the images for [Tbx1.sup.neo2/neo2] and [Foxn1.sup.1089/1089] hypoplastic lobes were duplicates. The correct figure part is below. The HTML and PDF files have been [...]

We report on 2 patients with compound heterozygous mutations in forkhead box N1 (FOXN1), a transcription factor essential for thymic epithelial cell (TEC) differentiation. TECs are critical for T cell development. Both patients had a presentation consistent with [T.sup.-/lo][B.sup.+][NK.sup.+] SCID, with normal hair and nails, distinct from the classic nude/SCID phenotype in individuals with autosomal-recessive FOXN1 mutations. To understand the basis of this phenotype and the effects of the mutations on FOXN1, we generated mice using CRISPR-Cas9 technology to genocopy mutations in 1 of the patients. The mice with the Foxn1 compound heterozygous mutations had thymic hypoplasia, causing a [T.sup.-][B.sup.+][NK.sup.+] SCID phenotype, whereas the hair and nails of these mice were normal. Characterization of the functional changes due to the Foxn1 mutations revealed a 5-amino acid segment at the end of the DNA-binding domain essential for the development of TECs but not keratinocytes. The transcriptional activity of this Foxn1 mutant was partly retained, indicating a region that specifies TEC functions. Analysis of an additional 9 FOXN1 mutations identified in multiple unrelated patients revealed distinct functional consequences contingent on the impact of the mutation on the DNA-binding and transactivation domains of FOXN1., Introduction The development of T cells expressing T cell receptors (TCRs) bearing a precise specificity for peptides embedded in self-HLA (MHC) molecules occurs in the thymus. At this site, T [...]

Abstract A statistically robust and biologically-based approach for analysis of microarray data is described that integrates independent biological knowledge and data with a global F-test for finding genes of interest that minimizes the need for replicates when used for hypothesis generation. First, each microarray is normalized to its noise level around zero. The microarray dataset is then globally adjusted by robust linear regression. Second, genes of interest that capture significant responses to experimental conditions are selected by finding those that express significantly higher variance than those expressing only technical variability. Clustering expression data and identifying expression-independent properties of genes of interest including upstream transcriptional regulatory elements (TREs), ontologies and networks or pathways organizes the data into a biologically meaningful system. We demonstrate that when the number of genes of interest is inconveniently large, identifying a subset of "beacon genes" representing the largest changes will identify pathways or networks altered by biological manipulation. The entire dataset is then used to complete the picture outlined by the "beacon genes." This allow construction of a structured model of a system that can generate biologically testable hypotheses. We illustrate this approach by comparing cells cultured on plastic or an extracellular matrix which organizes a dataset of over 2,000 genes of interest from a genome wide scan of transcription. The resulting model was confirmed by comparing the predicted pattern of TREs with experimental determination of active transcription factors.

To gain further insight into the basis for the extended longevity and delayed aging of Snell dwarf (dw/dw) mice, we have measured levels of expression of 2352 genes in liver of mice at 6 months of age. We find 60 genes for the which the Student's t statistic meets the arbitrary criterion of p < .001, and among these 17 meet the Bonferroni-adjusted significance criterion at p < .05, which corresponds to a nominal value of p < .00002. Using the Bonferroni criterion, we find that dwarf mice show increases in liver mRNA for two mannose-binding lectins, two DNA binding proteins, serum amyloid P component, corticosteroid-binding globulin, and insulin-like growth factor-binding protein 2, as well as decreases in a two phosphodiesterases, a pheromonebinding urinary protein, insulin-like growth factor-I (IGF-I), a calcium-binding protein calgranulin B, a deubiquitinating enzyme, a hydroxysteroid dehydrogenase, a DNA methyltransferase, a glycine transporter, and a placental lactogen. We also use this data set to compare the results of different suggested criteria for evaluating intergroup differences in gene expression. Of the 2352 genes examined, 524 (22%) showed a twofold difference between dwarf and normal mice, but most of these fail to meet the conventional significance criterion of p < .05, let alone criteria that have been adjusted to compensate for multiple comparison artifacts. The list of genes that show reliable differences between dwarf and control animals provides new insights into the range of changes induced by deficiencies in growth hormone, thyroid-stimulating hormone, and prolactin, and it will help to guide further studies of the pathways by which these hormone deficiencies contribute to delayed aging in these mutant mice.

Abstract Background Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression in the bladder target organ beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following chronic intravesical BCG therapy and to compare the results to non-specific pro inflammatory stimuli (LPS and TNF-α). For this purpose, C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-α. Seven days after the last instillation, the urothelium along with the submucosa was removed from detrusor muscle and the RNA was extracted from both layers for cDNA array experiments. Microarray results were normalized by a robust regression analysis and only genes with an expression above a conditional threshold of 0.001 (3SD above background) were selected for analysis. Next, genes presenting a 3-fold ratio in regard to the control group were entered in Ingenuity Pathway Analysis (IPA) for a comparative analysis in order to determine genes specifically regulated by BCG, TNF-α, and LPS. In addition, the transcriptome was precipitated with an antibody against RNA polymerase II and real-time polymerase chain reaction assay (Q-PCR) was used to confirm some of the BCG-specific transcripts. Results Molecular networks of treatment-specific genes generated several hypotheses regarding the mode of action of BCG. BCG-specific genes involved small GTPases and BCG-specific networks overlapped with the following canonical signaling pathways: axonal guidance, B cell receptor, aryl hydrocarbon receptor, IL-6, PPAR, Wnt/β-catenin, and cAMP. In addition, a specific detrusor network expressed a high degree of overlap with the development of the lymphatic system. Interestingly, TNF-α-specific networks overlapped with the following canonical signaling pathways: PPAR, death receptor, and apoptosis. Finally, LPS-specific networks overlapped with the LPS/IL-1 mediated inhibition of RXR. Because NF-kappaB occupied a central position in several networks, we further determined whether this transcription factor was part of the responses to BCG. Electrophoretic mobility shift assays confirmed the participation of NF-kappaB in the mouse bladder responses to BCG. In addition, BCG treatment of a human urothelial cancer cell line (J82) also increased the binding activity of NF-kappaB, as determined by precipitation of the chromatin by a NF-kappaB-p65 antibody and Q-PCR of genes bearing a NF-kappaB consensus sequence. Next, we tested the hypothesis of whether small GTPases such as LRG-47 are involved in the uptake of BCG by the bladder urothelium. Conclusion As expected, BCG treatment induces the transcription of genes belonging to common pro-inflammatory networks. However, BCG also induces unique genes belonging to molecular networks involved in axonal guidance and lymphatic system development within the bladder target organ. In addition, NF-kappaB seems to play a predominant role in the bladder responses to BCG therapy. Finally, in intact urothelium, BCG-GFP internalizes in LRG-47-positive vesicles. These results provide a molecular framework for the further study of the involvement of immune and nervous systems in the bladder responses to BCG therapy.