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Immunohistochemistry for ZBP-89 and Ki67 in WT and VC:Zfp148F/F mice

Percentage of APC, CTNNB1 and ZNF148 mutations in CRC (TCGA)

In colorectal cancer, APC-mediated induction of unregulated cell growth involves posttranslational mechanisms that prevent proteasomal degradation of proto-oncogene β-catenin (CTNNB1) and its eventual translocation to the nucleus. However, about 10% of colorectal tumors also exhibit increased CTNNB1 mRNA. Here, we show in colorectal cancer that increased expression of ZNF148, the gene coding for transcription factor ZBP-89, correlated with reduced patient survival. Tissue arrays showed that ZBP-89 protein was overexpressed in the early stages of colorectal cancer. Conditional deletion of Zfp148 in a mouse model of Apc-mediated intestinal polyps demonstrated that ZBP-89 was required for polyp formation due to induction of Ctnnb1 gene expression. Chromatin immunoprecipitation (ChIP) and EMSA identified a ZBP-89–binding site in the proximal promoter of CTNNB1. Reciprocally, siRNA-mediated reduction of CTNNB1 expression also decreased ZBP-89 protein. ChIP identified TCF DNA binding sites in the ZNF148 promoter through which Wnt signaling regulates ZNF148 gene expression. Suppression of either ZNF148 or CTNNB1 reduced colony formation in WNT-dependent, but not WNT-independent cell lines. Therefore, the increase in intracellular β-catenin protein initiated by APC mutations is sustained by ZBP-89–mediated feedforward induction of CTNNB1 mRNA. Cancer Res; 76(23); 6877–87. ©2016 AACR.

Supplemental Methods, Figure Legends and References for Supplemental Methods

ZBP-89 Probe Sets:BFCOL1 and BERF1

Critically ill and anesthetized patients commonly receive life-sustaining medications by pump-driven continuous intravenous infusion. Microinfusion refers to delivering concentrated drugs with low flow carriers to conserve fluid administration. Most infused medications are water-soluble. Delivery onset lag times have been identified for microinfusions of water-soluble drugs or experimental surrogates. Drugs may be formulated as emulsions. Initiation of emulsion microinfusions has not been described. We tested in vitro the hypothesis that an emulsion's physical characteristics would influence its microinfusion delivery onset. We adapted an established in vitro model of pump-driven continuous intravenous microinfusion to compare the delivery of methylene blue as a surrogate for water-soluble drugs and a 10% lipid emulsion as a surrogate for a drug formulated as an emulsion. The drug surrogates joined the carrier with carrier flow vertically upwards, vertically downwards or horizontally. We measured the times to 5%, 50% and 95% of plateau delivery. Emulsion entry into a vertical (upwards) carrier flow resulted in a rapid initial emulsion delivery exceeding predictions of delivery models. Emulsion entry into both horizontal and vertical (downwards) carrier flows resulted in long lag times to steady state. Methylene blue delivery was unaffected by carrier flow orientation. Initiating microinfusion emulsion delivery with upward flow can result in a relative bolus, whereas long delivery lags would be expected with horizontal or downwards flow. An emulsion might carry a high potency drug having significant physiologic effects, e. g. clevidipine. Unrecognized, differences in initial emulsion delivery kinetics depending on carrier flow orientation may have clinical implications for both efficacy and safety.

The impact of omeprazole (OM), a widely used over-the-counter proton pump inhibitor, on weight gain has not been extensively explored. We examined what factors, e.g., diet composition, microbiota, genetic strain, and sex, might affect weight gain in mice fed a high caloric diet while on OM. Inbred C57BL/6J strain, a 50:50 hybrid (B6SJLF1/J) strain, and mice on a highly mixed genetic background were fed four diets: standard chow (STD, 6% fat), STD with 200 ppm OM (STD + O), a high-energy chow (HiE, 11% fat), and HiE chow with OM (HiE + O) for 17 wk. Metabolic analysis, body composition, and fecal microbiota composition were analyzed in C57BL/6J mice. Oral glucose tolerance tests were performed using mice on the mixed background. After 8 wk, female and male C57BL/6J mice on the HiE diets ate less, whereas males on the HiE diets compared with the STD diets gained weight. All diet treatments reduced energy expenditure in females but in males only those on the HiE + O diet. Gut microbiota composition differed in the C57BL/6J females but not the males. Hybrid B6SJLF1/J mice showed similar weight gain on all test diets. In contrast, mixed strain male mice fed a HiE + O diet gained ∼40% more weight than females on the same diet. In addition to increased weight gain, mixed genetic mice on the HiE + O diet cleared glucose normally but secreted more insulin. We concluded that sex and genetic background define weight gain and metabolic responses of mice on high caloric diets and OM.

Intestinal metaplasia (IM) is a gastric cancer precursor lesion (GCPL) and an extremely high risk factor for progression to gastric cancer (GC). Clinical guidelines recommend that patients with extensive IM undergo a gastroscopy every 3 years. However, protein biomarkers that indicate a transition from IM to GC are lacking. Our group recently identified an interferon-alpha (IFN alpha)-responsive gene, Schlafen 4 (Slfn4), in immune cells that correlates with metaplastic changes in Helicobacter-infected mice. We therefore tested the hypothesis that a human homolog of Slfn4, namely, Schlafen 5 (SLFN5), correlates with progression of GCPL to GC. Jurkat T-lymphoid and HL-60 myeloid cell lines were treated with IFN alpha, and SLFN5 mRNA was quantified by quantitative PCR. SLFN5 protein expression in the inflamed gastric mucosa was co-localized to specific immune cell types by immunohistochemistry using CD20, CD2, and MAC2 antibodies. SLFN5 expression was also determined by immunohistochemistry in formalin-fixed paraffin-embedded samples from individuals with non-atrophic gastritis, atrophic gastritis, complete IM, incomplete IM, and GC, respectively. The IFN alpha treatment of Jurkat and HL-60 cells induced SLFN5 mRNA. SLFN5 protein was expressed mainly by T lymphocytes in inflamed gastric mucosa. The highest level of SLFN5 expression was observed in patients with IM that progressed to GC. Receiver operating characteristic curves demonstrated that correlating SLFN5 expression with the histologic diagnosis of IM significantly increased the probability of identifying patients who may progress to GC. In this study population, elevated SLFN5 protein expression in patients with IM correlated with progression to GC.

Laboratory data suggest that newly initiated drug infusions reach steady-state delivery after a significant time lag. Depending on drug and carrier flow rates and the infusion system's common volume, lag times may exceed 20 or more minutes, especially in the neonatal/pediatric critical care environment. This study tested the hypothesis that a computer-executed algorithm controlling infusion pumps in a coordinated fashion predictably hastens the achievement of the intended steady-state drug delivery in a model of neonatal/pediatric drug infusion.We constructed an in vitro model of neonatal/pediatric drug infusions through a pediatric 4-Fr central venous catheter at total system flows of 2 mL/h or 12 mL/h, representing a clinically relevant infusion range. Methylene blue served as the model infused drug for quantitative analysis. A novel algorithm, based on Taylor Dispersion Theory of fluid flow through tubes and executed by a computer, generated flow patterns that controlled and coordinated drug and carrier delivery by syringe pumps. We measured the time to achieve the intended steady-state drug delivery by conventional initiation of the drug infusion ("turning on the drug pump") and by algorithm-controlled infusion initiation.At 2 mL/h total system flow, application of the algorithm reduced the time to achieve half of the intended drug delivery rate (T50) from 17 minutes [17, 18] to 3 minutes [3, 3] (median, interquartile range). At 12 mL/h total system flow, application of the algorithm reduced T50 from 6 minutes [6, 7] to 3 minutes [3, 3] The bootstrapped median difference is -14 (95% confidence interval [CI], -16 to -12, adjusted P=.00192) for 2 mL/h flow and -3 (95% CI, -4 to -3, adjusted P=.02061) for 12 mL/h flow. Compared with conventional initiation, the additional fluid required by the algorithm-directed infusion was 0.43 and 1.03 mL for the low- and high-infusion rates, respectively.The output of infusion pumps can be predictably controlled and coordinated by a computer-executed algorithm in a model of neonatal/pediatric drug infusions. Application of an algorithm can reduce the time to achieve the intended rate of infused drug delivery with minimal incremental volume administration.

Summary Objective: To test the hypothesis that steady-state drug delivery by continuous infusion is predictably affected by a second drug infusion in the same lumen. Background: Clinicians commonly administer two drugs by continuous infusion through one central venous catheter lumen (co-infusion). To limit fluid delivery, low flow rate carriers transport concentrated drug solutions; a method called microinfusion. How microinfusion delivery of one drug is affected by a second drug infusion has not been explored. Methods: Two water-soluble dyes, tartrazine and erioglaucine, infused at 3 ml·h−1, modeled drug delivery through a four stopcock linear manifold and catheter lumen. A pump drove a carrier fluid (10 ml·h−1). After tartrazine reached steady-state delivery, erioglaucine entered downstream or upstream of the tartrazine infusion. Quantitative spectrophotometry measured dye delivery. Results: Starting erioglaucine’s infusion upstream of tartrazine’s entry caused a transient tartrazine bolus (duration 10 min, peak drug delivery 20% higher than target levels). Starting erioglaucine’s infusion downstream produced a similar amplitude, briefer, bolus. Stopping the erioglaucine infusion caused a transient reduction in tartrazine delivery. Measured delivery profiles were comparable to prediction models. Conclusions: We confirmed the hypothesis that delivery of one infused drug is transiently affected by starting or stopping a second drug infusion in the same line. The magnitude of the changes can be estimated quantitatively. The clinical impact depends on the drugs being co-infused and patient sensitivity, but could be clinically important; the findings have safety implications for infused medication delivery to critically ill or anesthetized children. We recommend minimizing infusion system dead volumes, connecting the most essential infusion(s) to the main fluid pathway as close as possible to the patient, and recognizing the potential for unintended alterations in delivery when multiple drugs co-infuse.

In colorectal cancer, APC-mediated induction of unregulated cell growth involves posttranslational mechanisms that prevent proteasomal degradation of proto-oncogene β-catenin (CTNNB1) and its eventual translocation to the nucleus. However, about 10% of colorectal tumors also exhibit increased CTNNB1 mRNA. Here, we show in colorectal cancer that increased expression of ZNF148, the gene coding for transcription factor ZBP-89, correlated with reduced patient survival. Tissue arrays showed that ZBP-89 protein was overexpressed in the early stages of colorectal cancer. Conditional deletion of Zfp148 in a mouse model of Apc-mediated intestinal polyps demonstrated that ZBP-89 was required for polyp formation due to induction of Ctnnb1 gene expression. Chromatin immunoprecipitation (ChIP) and EMSA identified a ZBP-89–binding site in the proximal promoter of CTNNB1. Reciprocally, siRNA-mediated reduction of CTNNB1 expression also decreased ZBP-89 protein. ChIP identified TCF DNA binding sites in the ZNF148 promoter through which Wnt signaling regulates ZNF148 gene expression. Suppression of either ZNF148 or CTNNB1 reduced colony formation in WNT-dependent, but not WNT-independent cell lines. Therefore, the increase in intracellular β-catenin protein initiated by APC mutations is sustained by ZBP-89–mediated feedforward induction of CTNNB1 mRNA. Cancer Res; 76(23); 6877–87. ©2016 AACR.

Intravenous drug infusion driven by syringe pumps may lead to substantial temporal lags in achieving steady-state delivery at target levels when using very low flow rates ("microinfusion"). This study evaluated computer algorithms for reducing temporal lags via coordinated control of drug and carrier flows.Novel computer control algorithms were developed based on mathematical models of fluid flow. Algorithm 1 controlled initiation of drug infusion and algorithm 2 controlled changes to ongoing steady-state infusions. These algorithms were tested in vitro and in vivo using typical high and low dead volume infusion system architectures. One syringe pump infused a carrier fluid and a second infused drug. Drug and carrier flowed together via a manifold through standard central venous catheters. Samples were collected in vitro for quantitative delivery analysis. Parameters including left ventricular max dP/dt were recorded in vivo.Regulation by algorithm 1 reduced delivery delay in vitro during infusion initiation by 69% (low dead volume) and 78% (high dead volume). Algorithmic control in vivo measuring % change in max dP/dt showed similar results (55% for low dead volume and 64% for high dead volume). Algorithm 2 yielded greater precision in matching the magnitude and timing of intended changes in vivo and in vitro.Compared with conventional methods, algorithm-based computer control of carrier and drug flows can improve drug delivery by pump-driven intravenous infusion to better match intent. For norepinephrine infusions, the amount of drug reaching the bloodstream per time appears to be a dominant factor in the hemodynamic response to infusion.

To test the hypothesis that steady-state drug delivery by continuous infusion is predictably affected by a second drug infusion in the same lumen.Clinicians commonly administer two drugs by continuous infusion through one central venous catheter lumen (co-infusion). To limit fluid delivery, low flow rate carriers transport concentrated drug solutions; a method called microinfusion. How microinfusion delivery of one drug is affected by a second drug infusion has not been explored.Two water-soluble dyes, tartrazine and erioglaucine, infused at 3 ml · h(-1), modeled drug delivery through a four stopcock linear manifold and catheter lumen. A pump drove a carrier fluid (10 ml · h(-1)). After tartrazine reached steady-state delivery, erioglaucine entered downstream or upstream of the tartrazine infusion. Quantitative spectrophotometry measured dye delivery.Starting erioglaucine's infusion upstream of tartrazine's entry caused a transient tartrazine bolus (duration 10 min, peak drug delivery 20% higher than target levels). Starting erioglaucine's infusion downstream produced a similar amplitude, briefer, bolus. Stopping the erioglaucine infusion caused a transient reduction in tartrazine delivery. Measured delivery profiles were comparable to prediction models.We confirmed the hypothesis that delivery of one infused drug is transiently affected by starting or stopping a second drug infusion in the same line. The magnitude of the changes can be estimated quantitatively. The clinical impact depends on the drugs being co-infused and patient sensitivity, but could be clinically important; the findings have safety implications for infused medication delivery to critically ill or anesthetized children. We recommend minimizing infusion system dead volumes, connecting the most essential infusion(s) to the main fluid pathway as close as possible to the patient, and recognizing the potential for unintended alterations in delivery when multiple drugs co-infuse.

of corticosterone level to baseline in 4 h after indomethacin administration. Indomethacininduced corticosterone rise was inhibited by NBI 27914 injection and these results confirm the critical role of CRF1 receptors in the activation of the HPA axis. Both astressin and astressin2-B injection resulted in a delay of the recovery to basal circulating corticosterone levels. The impaired termination of corticosterone response to indomethacin caused by astressin2-B was followed by aggravation of indomethacin-induced gastric injury. Conclusions: The results suggest that CRF injected into the periphery at the dose, which markedly increased plasma corticosterone levels, may induce protection against indomethacin-produced gastric injury through both CRF1 and CRF2 receptors. CRF2 receptor signaling pathway contributes to the appropriate termination of indomethacin-induced corticosterone secretion and the maintainance of gastric mucosal integrity during ulcerogenic action of indomethacin in rats. The study was supported by grants from RFBR N13-04-01680a and PRAS 5 and 7.