Novel ACE mutations mimicking sarcoidosis by increasing blood ACE levels.

An elevated blood angiotensin I-converting enzyme (ACE) supports diagnosis of sarcoidosis and Gaucher disease. However, some ACE mutations increase ACE shedding, and patients with these mutations are therefore at risk of being incorrectly diagnosed with sarcoidosis because of elevated serum ACE levels. We applied a novel approach called "ACE phenotyping" to identify possible ACE mutations in 3 pulmonary clinic patients that had suspected sarcoidosis based on elevated blood ACE levels. Conformational fingerprinting of ACE indicated that these mutations may be localized in the stalk region of the protein and these were confirmed by whole exome sequencing. Index patient 1 (IP1) had a mutation (P1199L) that had been previously identified, while the other 2 patients had novel ACE mutations. IP2 had 2 mutations, T887M and N1196K (eliminating a putative glycosylation site), while IP3 had a stop codon mutation Q1124X (eliminating the transmembrane anchor). We also performed a comprehensive analysis of the existing database of all ACE mutations to estimate the proportion of mutations increasing ACE shedding. The frequency of ACE mutations resulting in increased blood ACE levels may be much higher than previously estimated. ACE phenotyping, together with whole exome sequencing, is a diagnostic approach that could prevent unnecessary invasive and/or costly diagnostic procedures, or potentially harmful treatment for patients misdiagnosed on the basis of elevated blood ACE levels.

Induction of Antigen-Independent Proliferation of Regulatory T-Cells by TNF Superfamily Ligands OX40L and GITRL.

TNF receptor superfamily comprises many T-cell costimulatory receptors, including TNFRSF1, TNFRSF2, TNFRSF4 (OX40), TNFRSF9 (4-1BB), TNFRSF18 (GITR), and TNFRSF7 (CD27). Signaling through these costimulatory stimulatory receptors can promote conventional T-cell (Tconv) proliferation, and effector functions in an antigen-dependent manner. Thus, agonistic antibodies and ligands for OX40, 4-1BB, GITR, and CD27 have been tested for inducing T-cell-mediated antitumor responses in several cancers. However, recently emerging reports show critical role for TNFR signaling in regulatory T-cell (Treg) differentiation and expansion, which might suppress effector T-cell proliferation and functions. Here, we show preferential over expression of TNFR2, OX40, 4-1BB, and GITR in Treg cells over Tconv cells, and the ability of OX40L and GITRL to induce selective proliferation of Treg cells, but not Tconv cells, in an antigen-independent manner. We describe the standard protocols used for Affymetrix gene expression profiling, T-cell isolation, and Cell Trace Violet-based cell proliferation assay.

Correlative Analysis of miRNA Expression and Oncotype Dx Recurrence Score in Estrogen Receptor Positive Breast Carcinomas.

Altered expression of miRNAs has been observed in many types of cancer, including breast cancer, and shown to contribute to cancer growth, aggressiveness, and response to therapies. In this pilot study, we investigated the possible correlation of miRNAs with risk of recurrence of estrogen receptor positive, lymph node-negative mammary carcinomas as determined by the Oncotype DX® Breast Cancer assay. To accomplish this, we extracted RNA from a collection of breast carcinomas that had previously been analyzed by Oncotype DX®. Multiple Let-7 family members were negatively correlated with the recurrence score (RS), which is consistent with their tumor suppressor properties. Additional miRNAs were found to positively correlate with RS, including miR-377-5p, miR-633b, miR-548t and miR-3648. Pathway analysis of putative and validated targets suggests that these miRNAs may have a diverse range of functions that may contribute to tumor recurrence. Taken together, these findings provide evidence that a miRNA expression signature can be developed to aid existing methods to determine the risk of recurrence for women with estrogen receptor positive breast cancers treated with endocrine therapy.

Diagnostic SNPs reveal widespread introgressive hybridization between introduced bighead and silver carp in the Mississippi River Basin.

Hybridization among conspecifics in native and introduced habitats has important implications for biological invasions in new ecosystems. Bighead (Hypophthalmichthys nobilis) and silver carp (H. molitrix) are genetically isolated and occur in sympatry within their native range. Following their introduction to North America, however, introgressant hybrids have been reported throughout their expanded range within the Mississippi River Basin (MRB). The extent of introgression, both spatially and generationally, is largely unknown. Therefore, we examined mixed-species populations from across the MRB to characterize the extent of interspecific gene flow. We assayed 2798 individuals from nine locations with a suite of species-diagnostic SNPs (57 nuclear and one mitochondrial). Forty-four per cent (n = 1244) of individuals displayed hybrid genotypes. Moreover, the composition of hybrid genotypes varied among locations and represented complex hybrid swarms with multiple generations of gene flow. Introgressive hybrids were identified from all locations, were bidirectional and followed a bimodal distribution consisting primarily of parental or parental-like genotypes and phenotypes. All described hybrid categories were present among individuals from 1999 to 2008, with parents and later-generation backcrosses representing the largest proportion of individuals among years. Our mitochondrial SNP (COII), tested on a subset of 730 individuals, revealed a silver carp maternal bias in 13 of 21 (62%) F1 hybrids, in all silver carp backcrosses, and maintained throughout many of the bighead carp backcrosses. The application of this suite of diagnostic markers and the spatial coverage permits a deeper examination of the complexity in hybrid swarms between two invasive, introduced species.

Restriction site-associated DNA sequencing generates high-quality single nucleotide polymorphisms for assessing hybridization between bighead and silver carp in the United States and China.

Bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix) are invasive species and listed as US federally injurious species under the Lacy Act. They have established populations in much of the Mississippi River Basin (MRB; Mississippi, Illinois, and Missouri rivers) and are capable of producing fertile hybrids and complex introgression. Characterizing the composition of this admixture requires a large set of high-quality, evolutionarily conserved, diagnostic genetic markers to aid in the identification and management of these species in the midst of morphological ambiguity. Restriction site-associated DNA (RAD) sequencing of 45 barcoded bighead and silver carp from the United States and China produced reads that were aligned to the silver carp transcriptome yielded 261 candidate single nucleotide polymorphisms (SNPs) with fixed allelic differences between the two species. We selected the highest quality 112 SNP loci for validation using 194 putative pure-species and F1 hybrids from the MRB and putative bighead carp and silver carp pure species from China (Amur, Pearl and Yangtze rivers). Fifty SNPs were omitted due to design/amplification failure or lack of diagnostic utility. A total of 57 species-diagnostic SNPs conserved between carp species in US and Chinese rivers were identified; 32 were annotated to functional gene loci. Twenty-seven of the 181 (15%) putative pure species were identified as hybrid backcrosses after validation, including three backcrosses from the Amur River, where hybridization has not been documented previously. The 57 SNPs identified through RAD sequencing provide a diagnostic tool to detect population admixture and to identify hybrid and pure-species Asian carps in the United States and China.

Positional differences in the wound transcriptome of skin and oral mucosa.

BACKGROUND: When compared to skin, oral mucosal wounds heal rapidly and with reduced scar formation. Recent studies suggest that intrinsic differences in inflammation, growth factor production, levels of stem cells, and cellular proliferation capacity may underlie the exceptional healing that occurs in oral mucosa. The current study was designed to compare the transcriptomes of oral mucosal and skin wounds in order to identify critical differences in the healing response at these two sites using an unbiased approach. RESULTS: Using microarray analysis, we explored the differences in gene expression in skin and oral mucosal wound healing in a murine model of paired equivalent sized wounds. Samples were examined from days 0 to 10 and spanned all stages of the wound healing process. Using unwounded matched tissue as a control, filtering identified 1,479 probe sets in skin wounds yet only 502 probe sets in mucosal wounds that were significantly differentially expressed over time. Clusters of genes that showed similar patterns of expression were also identified in each wound type. Analysis of functionally related gene expression demonstrated dramatically different reactions to injury between skin and mucosal wounds. To explore whether site-specific differences might be derived from intrinsic differences in cellular responses at each site, we compared the response of isolated epithelial cells from skin and oral mucosa to a defined in vitro stimulus. When cytokine levels were measured, epithelial cells from skin produced significantly higher amounts of proinflammatory cytokines than cells from oral mucosa. CONCLUSIONS: The results provide the first detailed molecular profile of the site-specific differences in the genetic response to injury in mucosa and skin, and suggest the divergent reactions to injury may derive from intrinsic differences in the cellular responses at each site.

Silvestrol regulates G2/M checkpoint genes independent of p53 activity.

As previously reported, silvestrol, a rocaglate derivative isolated from Aglaia foveolata, has similar potency to paclitaxel and camptothecin against cultured human cancer cells. Furthermore, silvestrol can inhibit cancer cell growth in mice without noticeable toxicity when administered up to 5 mg/kg body weight (the highest dose tested). The purpose of the current study was to evaluate the mechanism of silvestrol's cytotoxicity in human prostate cancer cells (LNCaP). The molecular signature induced in LNCaP cells by silvestrol was evaluated using microarray analysis. The results revealed that 20 apoptosis and cell cycle related genes were significantly altered in LNCaP cells exposed to silvestrol. These included UBL-3, p21 and p300, which were up-regulated, and p53, which was down-regulated. Since p53 expression is governed primarily at the level of translation, p53 was also evaluated by Western blot. Silvestrol caused a dose-dependent decrease in p53 protein within 30 min of exposure with no p53 detectable after 6 h. Down-regulation of p53 by silvestrol was associated with down-regulation of MDM2 and not prevented by lactacystin suggesting that silvestrol-induced degradation of p53 is not mediated by the proteasome. A slight decrease in cyclin B was observed within 6 h of silvestrol exposure and phosphatase Cdc25C protein, which activates Cdc2, was also decreased. These data demonstrate that cytotoxicity induced by silvestrol in LNCaP cells is associated with a block in the cell cycle at the G2/M checkpoint and alterations in the expression of genes regulating apoptosis and cell cycle in a manner independent of p53.