An efficient noninvasive method for in vivo imaging of tumor oxygenation by using a low-field magnetic resonance scanner and a paramagnetic contrast agent is described. The methodology is based on Overhauser enhanced magnetic resonance imaging (OMRI), a functional imaging technique. OMRI experiments were performed on tumor-bearing mice (squamous cell carcinoma) by i.v. administration of the contrast agent Oxo63 (a highly derivatized triarylmethyl radical) at nontoxic doses in the range of 2-7 mmol/kg either as a bolus or as a continuous infusion. Spatially resolved pO 2 (oxygen concentration) images from OMRI experiments of tumor-bearing mice exhibited heterogeneous oxygenation profiles and revealed regions of hypoxia in tumors (<10 mmHg; 1 mmHg ‫؍‬ 133 Pa). Oxygenation of tumors was enhanced on carbogen (95% O 2͞5% CO2) inhalation. The pO2 measurements from OMRI were found to be in agreement with those obtained by independent polarographic measurements using a pO 2 Eppendorf electrode. This work illustrates that anatomically coregistered pO 2 maps of tumors can be readily obtained by combining the good anatomical resolution of water proton-based MRI, and the superior pO 2 sensitivity of EPR. OMRI affords the opportunity to perform noninvasive and repeated pO 2 measurements of the same animal with useful spatial (Ϸ1 mm) and temporal (2 min) resolution, making this method a powerful imaging modality for small animal research to understand tumor physiology and potentially for human applications.A bnormal values of pO 2 (the partial pressure of O 2 ) are linked to many pathophysiological conditions (e.g., ischemic diseases, reperfusion injury, and oxygen toxicity). Approximately one-third of human tumors evaluated for oxygen status have shown significant oxygen deficiency, and oxygen deficiency increases the tumor's resistance toward cancer treatment modalities, including radiation and chemotherapy (1, 2). Additionally, hypoxic microenvironments in tumors are known to promote processes driving malignant progression, such as angiogenesis, elimination of p53 tumor suppressor activity, genetic instability, and metastasis (3-5). Understanding of tumor hypoxia could lead to the discovery of diagnostic and prognostic markers for malignant progression, discovery of novel therapeutic targets, and the development of new constructs for gene therapy applications in human cancer. Hence, a noninvasive technique that could accurately and repetitively measure tissue oxygenation would find broad application in clinical and basic research. Unfortunately, the currently used electrochemical method (6) for in vivo oxygen measurement is an invasive technique applicable only to accessible tumors. Further, the technique is hampered by measurements of only a small part of the total tumor, which cannot be re-evaluated. Several magnetic resonance techniques (7, 8) have been developed for in vivo oximetry, including spin label oximetry (9), MRI (10), and electron paramagnetic resonance imaging (EPRI) (11,12). The blood oxygen level-dependent...

Transcription activator-like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-κB signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-beta and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-beta 2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, alpha-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-beta in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery.

Transforming growth factor-␤ (TGF-␤)-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. In this report, we identify GADD45b as an effector of TGF-␤-induced apoptosis. GADD45b has been shown to be a positive mediator of apoptosis induced by certain cytokines and oncogenes. We show that Gadd45b is an immediateearly response gene for TGF-␤ and that the proximal Gadd45b promoter is activated by TGF-␤ through the action of Smad2, Smad3, and Smad4. We show that ectopic expression of GADD45b in AML12 murine hepatocytes is sufficient to activate p38 and to trigger apoptotic cell death, whereas antisense inhibition of Gadd45b expression blocks TGF-␤-dependent p38 activation and apoptosis. Furthermore, we also show that TGF-␤ can activate p38 and induce apoptosis in mouse primary hepatocytes from wild-type mice, but not from Gadd45b ؊/؊ mice. All of these findings suggest that GADD45b participates in TGF-␤-induced apoptosis by acting upstream of p38 activation.