Direct effects of 2,3,7,8 tetrachlorodibenzo-p-dioxin on antigen-presenting cells and molecular signaling pathways in dendritic cells

In experimentally exposed mice, the environmental contaminant 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) produces significant suppression of adaptive immune responses at low doses. However, the underlying biochemical and cellular mechanisms of TCDD-induced immunotoxicity have remained elusive since the identification of these effects nearly 30 years ago. Antigen-presenting cells (APC) constitute various populations of cells essential for the initiation and maintenance of adaptive immune responses, and represent a potential target of TCDD toxicity. Thus, the studies presented here address the ability of TCDD to directly affect APC. The underlying objectives of these studies focus on the investigation of molecular signaling pathways and cellular processes potentially affected by TCDD. In order to eliminate conflicting variables found in vivo, we used ex vivo and in vitro models to address these objectives. Initial studies investigated the status and behavior of the aryl hydrocarbon receptor (AhR), a transcription factor recognized as the principal mediator of TCDD-induced immunotoxic effects, in the two main APC populations, macrophages and dendritic cells (DC). The results demonstrated that both APC populations expressed AhR. However, TCDD induced binding of AhR to dioxin response elements only in macrophages, and not DC. Because TCDD has been shown to alter DC function and survival in vivo, the possibility that TCDD altered other signaling pathways was addressed. Specifically, activation of the transcription factor NF-kB/Rel, integral in DC generation and function, was found to be suppressed by TCDD. This suppression was apparently mediated by a physical association between the AhR and proteins of NF-kB/Rel. Additional studies demonstrated that TCDD enhances the maturation of DC and appears to sensitize DC to apoptosis. These data establish that TCDD directly affects DC on the molecular and cellular levels and support several potential mechanisms of TCDD-induced immunotoxicity

Microneedles for Drug Delivery via the Gastrointestinal Tract

Both patients and physicians prefer the oral route of drug delivery. The gastrointestinal (GI) tract, though, limits the bioavailability of certain therapeutics because of its protease and bacteria-rich environment as well as general pH variability from pH 1 to 7. These extreme environments make oral delivery particularly challenging for the biologic class of therapeutics. Here, we demonstrate proof-of-concept experiments in swine that microneedle-based delivery has the capacity for improved bioavailability of a biologically active macromolecule. Moreover, we show that microneedle-containing devices can be passed and excreted from the GI tract safely. These findings strongly support the success of implementation of microneedle technology for use in the GI tract.National Institutes of Health (U.S.) (Grant EB000244)National Institutes of Health (U.S.) (Grant T32DK7191-38-S1

Inhibition of p70 S6 Kinase (S6K1) Activity by A77 1726 and Its Effect on Cell Proliferation and Cell Cycle Progress

AbstractLeflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor–mediated signaling pathway. In vitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 μM, respectively. Exogenous uridine partially blocked A77 1726–induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726–induced cell cycle arrest in the S phase

Feed-Forward Microprocessing and Splicing Activities at a MicroRNA–Containing Intron

The majority of mammalian microRNA (miRNA) genes reside within introns of protein-encoding and non-coding genes, yet the mechanisms coordinating primary transcript processing into both mature miRNA and spliced mRNA are poorly understood. Analysis of melanoma invasion suppressor miR-211 expressed from intron 6 of melastatin revealed that microprocessing of miR-211 promotes splicing of the exon 6–exon 7 junction of melastatin by a mechanism requiring the RNase III activity of Drosha. Additionally, mutations in the 5′ splice site (5′SS), but not in the 3′SS, branch point, or polypyrimidine tract of intron 6 reduced miR-211 biogenesis and Drosha recruitment to intron 6, indicating that 5′SS recognition by the spliceosome promotes microprocessing of miR-211. Globally, knockdown of U1 splicing factors reduced intronic miRNA expression. Our data demonstrate novel mutually-cooperative microprocessing and splicing activities at an intronic miRNA locus and suggest that the initiation of spliceosome assembly may promote microprocessing of intronic miRNAs

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Development of an Open Education Resource for Pharmacology to Address Equity in Veterinary Medicine Instruction

Open education resources (OERs), an alternative to expensive higher education textbooks, are a potentially valuable tool to both bring equity to the college classroom and enhance learning. To determine if an OER can be designed to optimize learning and provide equity to the student in a veterinary pharmacology classroom, an OER content outline and sample chapter were designed and developed. In addition to the cost saving benefit of the OER, it was determined that the addition of notable pharmacology role model profiles that “look like me” to the OER would be a viable means to improve the self-efficacy of women and underrepresented students and support retention in the field. Universities have recognized the need to reduce textbook costs and have put into place tools and programs to facilitate building OERs. Using software like PressBooks and other resources to aid in textbook design, a three part, eight chapter OER outline with chapter content description was produced, and a completed chapter, as an example of how the content and learning and equity elements would be integrated. The successful development of these two products was not difficult and with the proper instruction and support a completed OER to better convey the principles of pharmacology in a veterinary setting and bring equity to low income, women and underrepresented students could be produced. The products of this project will serve as the foundation to a completed and applicable OER, and at that time the true impact of the OER can be fully understood