To report the first clinical experience with targeted vessel ablation during magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) treatment of symptomatic uterine fibroids.

Great efforts have been made to predict disease behavior over time and the response to treatment in Crohn's disease (CD). Such understanding could personalize therapy. Early introduction of more aggressive therapies to patients at high risk and no introduction of predictable refractory treatments could become possible. We hence tested the influence of the NOD2 carrier status on treatment response.

Analysis of cellular signaling networks typically involves targeted measurements of phosphorylated protein intermediates. However, phosphoproteomic analyses usually require affinity enrichment of phosphopeptides and can be complicated by artifactual changes in phosphorylation caused by uncontrolled preanalytical variables, particularly in the analysis of tissue specimens. We asked whether changes in protein expression, which are more stable and easily analyzed, could reflect network stimulation and inhibition. We employed this approach to analyze stimulation and inhibition of the epidermal growth factor receptor (EGFR) by EGF and selective EGFR inhibitors. Shotgun analysis of proteomes from proliferating A431 cells, EGF-stimulated cells, and cells co-treated with the EGFR inhibitors cetuximab or gefitinib identified groups of differentially expressed proteins. Comparisons of these protein groups identified 13 proteins whose EGF-induced expression changes were reversed by both EGFR inhibitors. Targeted multiple reaction monitoring analysis verified differential expression of 12 of these proteins, which comprise a candidate EGFR inhibition signature. We then tested these 12 proteins by multiple reaction monitoring analysis in three other models: 1) a comparison of DiFi (EGFR inhibitor-sensitive) and HCT116 (EGFR-insensitive) cell lines, 2) in formalin-fixed, paraffin-embedded mouse xenograft DiFi and HCT116 tumors, and 3) in tissue biopsies from a patient with the gastric hyperproliferative disorder Ménétrier's disease who was treated with cetuximab. Of the proteins in the candidate signature, a core group, including c-Jun, Jagged-1, and Claudin 4, were decreased by EGFR inhibitors in all three models. Although the goal of these studies was not to validate a clinically useful EGFR inhibition signature, the results confirm the hypothesis that clinically used EGFR inhibitors generate characteristic protein expression changes. This work further outlines a prototypical approach to derive and test protein expression signatures for drug action on signaling networks.

IGF-I-stimulated sarcoma viral oncogene (Src) activation during hyperglycemia is required for propagating downstream signaling. The aim of the current study was to determine the mechanism by which hyperglycemia enhances IGF-I-stimulated Src activation and the role of NADPH oxidase 4 (Nox4) and protein kinase C ζ (PKCζ) in mediating this response in vascular smooth muscle cells (VSMCs). Nox4 expression was analyzed in VSMCs exposed to hyperglycemia. The role of Nox4-derived reactive oxygen species (ROS) in IGF-I-stimulated Src activation was investigated via knockdown of Nox4. Different isoforms of PKC were screened to investigate their role in hyperglycemia-induced Nox4. The oxidation of Src was shown to be a prerequisite for its activation in response to IGF-I during hyperglycemia. Hyperglycemia induced Nox4, but not Nox1, and p22 phagocyte oxidase (p22phox) expression and IGF-I stimulated Nox4/p22phox complex formation, leading to increased ROS generation. Knockdown of Nox4 prevented ROS generation and impaired the oxidation and activation of Src in response to IGF-I, whereas knockdown of Nox1 had no effect. PKCζ was shown to mediate the hyperglycemia-induced increase in Nox4 expression. The key observations in cultured VSMCs were confirmed in the diabetic mice. Nox4-derived ROS is responsible for the enhancing effect of hyperglycemia on IGF-I-stimulated Src activation, which in turn amplifies IGF-I-linked downstream signaling and biological actions.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) has a unique tight binding to dNTP substrates. Structural modeling of Ala-114 of HIV-1 RT suggests that longer side chains at this residue can reduce the space normally occupied by the sugar moiety of an incoming dNTP. Indeed, mutations at Ala-114 decrease the ability of RT to synthesize DNA at low dNTP concentrations and reduce the dNTP-binding affinity (K(d)) of RT. However, the K(d) values of WT and A114C RT remained equivalent with an acyclic dNTP substrate. Finally, mutant A114 RT HIV-1 vectors displayed a greatly reduced transduction in nondividing human lung fibroblasts (HLFs), while WT HIV-1 vector efficiently transduced both dividing and nondividing HLFs. Together these data support that the A114 residue of HIV-1 RT plays a key mechanistic role in the dNTP binding of HIV-1 RT and the unique viral infectivity of target cell types with low dNTP pools.

A radiation-induced blood-brain barrier (BBB) breakdown has been supposed to explain the acute radiation syndrome and the delayed brain radiation injury, but it has been clearly demonstrated only at high doses. In a previous study (Diserbo et al., 2002), we showed that non-lethal total body irradiation produced an early transient increase in BBB permeability in rats but the underlying mechanisms of radiation-induced BBB breakdown remain unclear. In the present work, the effects of ionizing radiation were studied on an in vitro BBB model. Gamma irradiation induced an increase in [(14)C]-sucrose BBB permeability that can be detected 72 h after exposure at doses up to 4 Gy. This increase was more important 8 days after irradiation and could be limited by dexamethasone treatment. An increase in fluorescein and FITC-dextrans (4 kDa/70 kDa) permeability was also observed, which can be related to a substantial opening of endothelial cell tight-junctions but without massive modification of tight-junction protein (ZO-1, ZO-2, claudin-5, occludin) immunolabeling even 8 days after 25 Gy exposure. Formation of actin stress fibers occurred in endothelial cells 8 days after 25 Gy exposure. A progressive decrease in cellular density associated with a simultaneous spreading of the endothelial cells was also observed after irradiation. Anti-γH2AX immunolabeling was used to investigate both DNA double-strand break induction and repair rates in endothelial cells. It revealed long-lasting DNA double-strand breaks after gamma irradiation. A better understanding and awareness of these phenomena are essential for designing appropriate pharmacotherapy in radiation-therapy and treatment of accidental overexposure.

Biomaterials are increasingly being investigated as a means of reducing stress within the ventricular wall of infarcted hearts and thus attenuating pathological remodelling and loss of function. In this context, we have examined the influence of timing of delivery on the efficacy of a polyethylene glycol hydrogel polymerised with an enzymatically degradable peptide sequence. Delivery of the hydrogel immediately after infarct induction resulted in no observable improvements, but a delay of one week in delivery resulted in significant increases in scar thickness and fractional shortening, as well as reduction in end-systolic diameter against saline controls and immediately injected hydrogel at both 2 and 4 weeks post-infarction (p < 0.05). Hydrogels injected at one week were degraded significantly slower than those injected immediately and this may have played a role in the differing outcomes. The hydrogel assumed markedly different morphologies at the two time points having either a fibrillar or bulky appearance after injection immediately or one week post-infarction respectively. We argue that the different morphologies result from infarction induced changes in the cardiac structure and influence the degradability of the injectates. The results indicate that timing of delivery is important and that very early time points may not be beneficial.

The melastatin transient receptor potential (TRP) channel, TRPM4, is a critical regulator of smooth muscle membrane potential and arterial tone. Activation of the channel is Ca(2+)-dependent, but prolonged exposures to high global Ca(2+) causes rapid inactivation under conventional whole-cell patch clamp conditions. Using amphotericin B perforated whole cell patch clamp electrophysiology, which minimally disrupts cytosolic Ca(2+) dynamics, we recently showed that Ca(2+) released from 1,2,5-triphosphate receptors (IP(3)R) on the sarcoplasmic reticulum (SR) activates TRPM4 channels, producing sustained transient inward cation currents (TICCs). Thus, Ca(2+)-dependent inactivation of TRPM4 may not be inherent to the channel itself but rather is a result of the recording conditions. We hypothesized that under conventional whole-cell configurations, loss of intrinsic cytosolic Ca(2+) buffering following cell dialysis contributes to inactivation of TRPM4 channels. With the inclusion of the Ca(2+) buffers ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, 10mM) or bis-ethane-N,N,N',N'-tetraacetic acid (BAPTA, 0.1mM) in the pipette solution, we mimic endogenous Ca(2+) buffering and record novel, sustained whole-cell TICC activity from freshly-isolated cerebral artery myocytes. Biophysical properties of TICCs recorded under perforated and whole-cell patch clamp were nearly identical. Furthermore, whole-cell TICC activity was reduced by the selective TRPM4 inhibitor, 9-phenanthrol, and by siRNA-mediated knockdown of TRPM4. When a higher concentration (10mM) of BAPTA was included in the pipette solution, TICC activity was disrupted, suggesting that TRPM4 channels on the plasma membrane and IP(3)R on the SR are closely opposed but not physically coupled, and that endogenous Ca(2+) buffer proteins play a critical role in maintaining TRPM4 channel activity in native cerebral artery smooth muscle cells.

Oxidative stress has been linked to noise- and drug-induced as well as age-related hearing loss. Antioxidants can attenuate the decline of cochlear structure and function after exposure to noise or drugs, but it is debated as to whether they can protect from age-related hearing loss. In a long-term longitudinal study, 10-month-old female CBA/J mice were placed on either a control or antioxidant-enriched diet and monitored through 24 months of age. Supplementation with vitamins A, C, and E, L-carnitine, and α-lipoic acid significantly increased the antioxidant capacity of inner ear tissues. However, by 24 months of age, the magnitude of hearing loss was equal between the two groups. Likewise, there were no significant differences in hair cell loss or degeneration of spiral ganglion cells. We conclude that dietary manipulations can alter cochlear antioxidant capacity but do not ameliorate age-related sensorineural hearing loss in the CBA/J mouse.

When pregnant adolescent sheep are overnourished during pregnancy normal nutrient partitioning priorities to the gravid uterus are altered, leading to impaired placental development and fetal growth restriction. We hypothesized that decreasing dietary intake in overnourished dams during the final third of gestation may reverse this inappropriate nutrient partitioning in favor of the fetus. Adolescent ewes were offered control (C; n = 12) or high (H; n = 20) dietary intakes to induce normal vs. compromised placental development. Ten ewes receiving the H intake were switched to a low intake at d90 of gestation (HL). Between d90 to 130, HL dams lost weight and adiposity, and metabolic hormones and glucose at d130 were less than H and similar to C. In spite of these maternal changes, at d130 fetal bodyweight was equivalent in HL and H groups and ∼20% less than in C. A greater degree of brain sparing was evident in HL fetuses and glucose and insulin concentrations were more perturbed than in H fetuses. Relative to C, placentome weight was reduced by 46 and 32% in H and HL and the fetal:placentome weight ratio was H > HL > C. Placental vascular morphology was largely unaffected by maternal diet during late gestation but mRNA expression of five angiogenic genes was up-regulated in the fetal cotyledon of HL pregnancies, commensurate with blood vessel remodeling. Nevertheless, overfeeding to promote maternal anabolic growth during adolescent pregnancy impairs feto-placental development that cannot be rescued by reducing maternal intake during the final third of gestation.

An efficient means of identifying protein biomarkers is essential to proper cancer management. A well-characterized proteome resource holds special promise for the discovery of novel biomarkers. However, quantification of the differences between physiological conditions together with deep down profiling has become increasingly challenging in proteomics. Here, we perform expression profiling of the colorectal cancer (CRC) proteome by stable isotope labeling and mass spectrometry. Quantitative analysis included performing mTRAQ and cICAT labeling in a pooled sample of three microsatellite stable (MSS) type CRC tissues and a pooled sample of their matched normal tissues. We identified and quantified a total of 3688 proteins. Among them, 1487 proteins were expressed differentially between normal and cancer tissues by higher than 2-fold; 1009 proteins showed increased expression in cancer tissue, whereas 478 proteins showed decreased expression. Bioinformatic analysis revealed that our data were largely consistent with known CRC relevant signaling pathways, such as the Wnt/β-catenin, caveolar-mediated endocytosis, and RAN signaling pathways. Mitochondrial dysfunction, known as the Waburg hypothesis, was also confirmed. Therefore, our data showing alterations in the proteomic profile of CRC constitutes a useful resource that may provide insights into tumor progression with later goal of identifying biologically and clinically relevant marker proteins. This article is part of a Special Issue entitled: Proteomics: The clinical link.

The rapid (2 min) nongenomic effects of aldosterone (ALDO) and/or spironolactone (MR antagonist), RU 486 (GR antagonist), atrial natriuretic peptide (ANP) and dimethyl-BAPTA (BAPTA) on the intracellular pH recovery rate (pHirr) via NHE1 (basolateral Na⁺/H⁺ exchanger isoform), after the acid load induced by NH₄Cl, and on the cytosolic free calcium concentration ([Ca²⁺](i)) were investigated in the proximal S3 segment isolated from rats, by the probes BCECF-AM and FLUO-4-AM, respectively. The basal pHi was 7.15±0.008 and the basal pHirr was 0.195±0.012 pH units/min (number of tubules/number of tubular areas=16/96). Our results confirmed the rapid biphasic effect of ALDO on NHE1: ALDO (10⁻¹² M) increases the pHirr to approximately 59% of control value, and ALDO (10⁻⁶ M) decreases it to approximately 49%. Spironolactone did not change these effects, but RU 486 inhibited the stimulatory effect and maintained the inhibitory effect. ANP (10⁻⁶ M) or BAPTA (5×10⁻⁵ M) alone had no significant effect on NHE1 but prevented both effects of ALDO on this exchanger. The basal [Ca²⁺](i) was 104±3 nM (15), and ALDO (10⁻¹² or 10⁻⁶ M) increased the basal [Ca²⁺](i) to approximately 50% or 124%, respectively. RU 486, ANP and BAPTA decreased the [Ca²⁺](i) and inhibited the stimulatory effect of both doses of ALDO. The results suggest the involvement of GR on the nongenomic effects of ALDO and indicate a pHirr-regulating role for [Ca²⁺](i) that is mediated by NHE1, stimulated/impaired by ALDO, and affected by ANP or BAPTA with ALDO. The observed nongenomic hormonal interaction in the S3 segment may represent a rapid and physiologically relevant regulatory mechanism in the intact animal under conditions of volume alterations.

Regrowth of peripheral spiral ganglion neuron (SGN) fibers is a primary objective in efforts to improve cochlear implant outcomes and to potentially reinnervate regenerated hair cells. Cyclic adenosine monophosphate (cAMP) regulates neurite growth and guidance via activation of protein kinase A (PKA) and Exchange Protein directly Activated by Cylic AMP (Epac). Here we explored the effects of cAMP signaling on SGN neurite length in vitro. We find that the cAMP analog, cpt-cAMP, exerts a biphasic effect on neurite length; increasing length at lower concentrations and reducing length at higher concentrations. This biphasic response occurs in cultures plated on laminin, fibronectin, or tenascin C suggesting that it is not substrate dependent. cpt-cAMP also reduces SGN neurite branching. The Epac-specific agonist, 8-pCPT-2'-O-Me-cAMP, does not alter SGN neurite length. Constitutively active PKA isoforms strongly inhibit SGN neurite length similar to higher levels of cAMP. Chronic membrane depolarization activates PKA in SGNs and also inhibits SGN neurite length. However, inhibition of PKA fails to rescue neurite length in depolarized cultures implying that activation of PKA is not necessary for the inhibition of SGN neurite length by chronic depolarization. Expression of constitutively active phosphatidylinositol 3-kinase, but not c-Jun N-terminal kinase, isoforms partially rescues SGN neurite length in the presence of activated PKA. Taken together, these results suggest that activation of cAMP/PKA represents a potential strategy to enhance SGN fiber elongation following deafness; however such therapies will likely require careful titration so as to promote rather than inhibit nerve fiber regeneration.

Cartilage oligomeric matrix protein (COMP), a secreted glycoprotein synthesized by chondrocytes, regulates proliferation and type II collagen assembly. Mutations in the COMP gene cause pseudoachondroplasia and multiple epiphyseal dysplasia. Previously, we have shown that expression of D469del-COMP in transgenic mice causes intracellular retention of D469del-COMP, thereby recapitulating pseudoachondroplasia chondrocyte pathology. This inducible transgenic D469del-COMP mouse is the only in vivo model to replicate the critical cellular and clinical features of pseudoachondroplasia. Here, we report developmental studies of D469del-COMP-induced chondrocyte pathology from the prenatal period to adolescence. D469del-COMP retention was limited prenatally and did not negatively affect the growth plate until 3 weeks after birth. Results of immunostaining, transcriptome analysis, and qRT-PCR suggest a molecular model in which D469del-COMP triggers apoptosis during the first postnatal week. By 3 weeks (when most chondrocytes are retaining D469del-COMP), inflammation, oxidative stress, and DNA damage contribute to chondrocyte cell death by necroptosis. Importantly, by crossing the D469del-COMP mouse onto a Chop null background (Ddit3 null), thereby eliminating Chop, the unfolded protein response was disrupted, thus alleviating both D469del-COMP intracellular retention and premature chondrocyte cell death. Chop therefore plays a significant role in processes that mediate D469del-COMP retention. Taken together, these results suggest that there may be an optimal window before the induction of significant D469del-COMP retention during which endoplasmic reticulum stress could be targeted.

Recent analytical advancements allow for large-scale glycomics and glycan-biomarker research with N-glycans released from complex protein mixtures of e.g. plasma with a wide range of protein concentrations. Protein enrichment techniques to obtain samples with a better representation of low-abundance proteins are hardy applied. In this study, hexapeptide ligands previously described for enrichment of low-abundance proteins in proteomics are evaluated for glycan analysis. A repeatable on-bead glycan release strategy was developed, and glycans were analyzed using capillary sieving electrophoresis on a DNA analyzer. Binding of proteins to the hexapeptide library occurred via the protein backbone. At neutral pH no discrimination between protein glycoforms was observed. Interestingly, glycan profiles of plasma with and without hexapeptide library enrichment revealed very similar patterns, despite the vast changes in protein concentrations in the samples. The most significant differences in glycosylation profiles were ascribed to a reduction in immunoglobulin-derived glycans. These results suggest that specific and sensitive biomarkers will be hard to access on the full plasma level using protein enrichment in combination with glycan analysis. Instead, fractionation techniques or profiling strategies on the glycopeptide level after enrichment are proposed for in-depth glycoproteomics research.

Elevated levels of survivin and histone deacetylases (HDACs) are often found over-expressed in human cancers, including colorectal cancer, and have been implicated in tumorigenesis. HDAC inhibition induces growth arrest and cell death in various transformed cell; however, the mechanisms by which this reduces cell viability in colorectal cancer cells remain unexplained.

Perturbation of energy homeostasis in skeletal muscle and liver resulting from a transient inhibition of mitochondrial energy transduction can produce effects of relevance for the control of hyperglycemia through activation of the AMP-activated protein kinase, as exemplified by the antidiabetic drug metformin. The present study focuses on uncoupling of oxidative phosphorylation rather than its inhibition as a trigger for such effects.

Fine epitope mapping of EF13D, a highly potent neutralizing monoclonal antibody specific for the anthrax edema factor (EF), was accomplished through random mutagenesis and yeast surface display. A yeast-displayed library of single point mutants of an EF domain III (DIII), comprising amino acids 624-800, was constructed by random mutagenesis and screened for reduced binding to EF13D. With this method, residues Leu 667, Ser 668, Arg 671, and Arg 672 were identified as key residues important for EF13D binding. They form a contiguous patch on a solvent-exposed surface at one end of the four-helix bundle of DIII. Computational protein-protein docking experiments between anEF13D model and a crystal structure of EF indicate that the EF13D heavy chain complementarity-determining region 3 (HCDR3) is deeply buried within a hydrophobic cleft between two helices of DIII and interacts directly with residues Leu 667, Ser 668, Arg 671 and Arg 672, providing an explanation for the high binding affinity. In addition, they show that the HCDR3 binding site overlaps with the binding site of the N-terminal lobe of calmodulin (CaM), an EF enzymatic activator, consistent with a previous finding showing direct competition with CaM that results in neutralization of EF. Identifying the neutralization epitope of EF13D on EF improves our understanding of the neutralization mechanism and has implications for vaccine development.

We used functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) to study the negative blood oxygenation level dependent (BOLD) signal and its underlying blood flow changes in healthy human subjects. This was combined with psychophysiological measurements to test that the negative BOLD signal is associated with functional inhibition. Electrical stimulation of the median nerve at 7 Hz evoked robust negative BOLD signals in the primary somatosensory cortex (SI) ipsilateral to stimulation, and positive BOLD signals in contralateral SI. The negative BOLD signal in ipsilateral SI was accompanied by commensurate decreases in relative regional cerebral blood flow (rCBF). Conjunction analysis of the fMRI and PET data revealed a region in the ipsilateral postcentral gyrus showing overlap of negative BOLD signals and relative rCBF decreases. The current perception threshold (CPT) at the ipsilateral finger during concomitant stimulation of the contralateral median nerve increased significantly, suggesting augmented functional inhibition. Since the CPT in the ipsilateral hallux did not significantly change in response to median nerve stimulation, it is more likely that the CPT-increase for the finger is due to functional inhibition (Kastrup et al., 2008) than to changes in selective attention. In conclusion, our data provide evidence that stimulus-induced reductions in relative rCBF may underlie the negative BOLD signal, which in turn may reflect increments in functional inhibition.

Chronic administration of high doses of hexavalent chromium [Cr(VI)] as sodium dichromate dihydrate (SDD) elicits alimentary cancers in mice. To further elucidate key events underlying tumor formation, a 90-day drinking water study was conducted in B6C3F1 mice. Differential gene expression was examined in duodenal and jejunal epithelial samples following 7 or 90days of exposure to 0, 0.3, 4, 14, 60, 170 or 520mg/L SDD in drinking water. Genome-wide microarray analyses identified 6562 duodenal and 4448 jejunal unique differentially expressed genes at day 8, and 4630 and 4845 unique changes, respectively, in the duodenum and jejunum at day 91. Comparative analysis identified significant overlap in duodenal and jejunal differential gene expression. Automated dose-response modeling identified >80% of the differentially expressed genes exhibited sigmoidal dose-response curves with EC(50) values ranging from 10 to 100mg/L SDD. Only 16 genes satisfying the dose-dependent differential expression criteria had EC(50) values <10mg/L SDD, 3 of which were regulated by Nrf2, suggesting oxidative stress in response to SDD at low concentrations. Analyses of differentially expressed genes identified over-represented functions associated with oxidative stress, cell cycle, lipid metabolism, and immune responses consistent with the reported effects on redox status and histopathology at corresponding SDD drinking water concentrations. Collectively, these data are consistent with a mode of action involving oxidative stress and cytotoxicity as early key events. This suggests that the tumorigenic effects of chronic Cr(VI) oral exposure likely require chronic tissue damage and compensatory epithelial cell proliferation.