Intravenous administration could become a delivery route of choice for prophylactic and curative gene therapies on condition that genes cross the capillary barrier and reach target tissues without being degraded. We investigated the kinetics and process of transgene delivery through mouse lung capillaries following DNA complexation with linear polyethylenimine (L-PEI) and intravenous injection. Using digoxin-labeled DNA we followed the cellular localization of DNA at different times after injection and correlated these findings with cell markers and transgene expression. At 2 h after injection some DNA was still localized on the interior of the capillary lumen, but other complexes had already crossed the barrier and resulted in gene expression. At 24 h after injection most

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Thyroid hormones (TH) are essential regulators of vertebrate development and metabolism. Central mechanisms governing their production have evolved, with the beta-TH receptor (TRbeta) playing a key regulatory role in the negative feedback effects of circulating TH levels on production of hypothalamic TRH and hypophyseal TSH. Both TRbeta-isoforms (TRbeta1 and TRbeta2) are expressed in the hypothalamus and pituitary. However, their respective roles in TH-dependent transcriptional regulation of TRH are undefined. We confirmed the preferential role of TRbeta vs. TRalpha isoforms in TRH regulation in wild-type mice in vivo by using the TRbeta preferential agonist GC-1. We next determined the effects of tissue-specific rescue of TRbeta1 and TRbeta2 isoforms by somatic gene transfer in hypothalami of TRbeta null (TRbeta(-/-)) mice. TH-dependent TRH transcriptional repression was impaired in TRbeta(-/-) mice, but was restored by cotransfection of either TRbeta1 or TRbeta2 into the hypothalamus. TRbeta1, but not TRbeta2, displayed a role in ligand-independent activation. In situ hybridization was used to examine endogenous TRH expression in the paraventricular nucleus of the hypothalamus of TRbeta(-/-) or TRalpha null (TRalpha(o/o)) mice under different thyroid states. In contrast to published data on TRbeta2(-/-) mice, we found that both ligand-independent TRH activation and ligand-dependent TRH repression were severely impaired in TRbeta(-/-) mice. This study thus provides functional in vivo data showing that both TRbeta1 and TRbeta2 isoforms have specific roles in regulating TRH transcription.

The toxic metalloid arsenic causes widespread misfolding and aggregation of cellular proteins. How these protein aggregates are formed in vivo, the mechanisms by which they affect cells, and how cells prevent their accumulation is not fully understood. To find components involved in these processes, we performed a genome-wide imaging screen and identified yeast deletion mutants with either enhanced or reduced protein aggregation levels during arsenite exposure. We show that many of the identified factors are crucial to safeguard protein homeostasis (proteostasis) and to protect cells against arsenite toxicity. The hits were enriched for various functions including protein biosynthesis and transcription, and dedicated follow-up experiments highlight the importance of accurate transcriptional and translational control for mitigating protein aggregation and toxicity during arsenite stress. Some of the hits are associated with pathological conditions, suggesting that arsenite-induced protein aggregation may affect disease processes. The broad network of cellular systems that impinge on proteostasis during arsenic stress identified in this current study provides a valuable resource and a framework for further elucidation of the mechanistic details of metalloid toxicity and pathogenesis.