A tale of two functions: enzymatic activity and translational repression by carboxyltransferase

Acetyl-CoA Carboxylase catalyzes the first committed step in fatty acid synthesis. Escherichia coli acetyl-CoA carboxylase is composed of biotin carboxylase, carboxyltransferase and biotin carboxyl carrier protein functions. The accA and accD genes that code for the α- and β-subunits, respectively, are not in an operon, yet yield an α2β2 carboxyltransferase. Here, we report that carboxyltransferase regulates its own translation by binding the mRNA encoding its subunits. This interaction is mediated by a zinc finger on the β-subunit; mutation of the four cysteines to alanine diminished nucleic acid binding and catalytic activity. Carboxyltransferase binds the coding regions of both subunit mRNAs and inhibits translation, an inhibition that is relieved by the substrate acetyl-CoA. mRNA binding reciprocally inhibits catalytic activity. Preferential binding of carboxyltransferase to RNA in situ was shown using fluorescence resonance energy transfer. We propose an unusual regulatory mechanism by which carboxyltransferase acts as a ‘dimmer switch’ to regulate protein production and catalytic activity, while sensing the metabolic state of the cell through acetyl-CoA concentration

Feasibility study of an internally cooled bipolar applicator for RF coagulation of hepatic tissue: Experimental and computational study

Purpose: To study the capacity of an internally cooled radiofrequency (RF) bipolar applicator to create sufficiently deep thermal lesions in hepatic tissue. Materials and methods: Three complementary methodologies were employed to check the electrical and thermal behaviour of the applicator under test. The experimental studies were based on excised bovine (ex vivo study) and porcine liver (in vivo study) and the theoretical models were solved by means of the finite element method (FEM). Results: Experimental and computational results showed good agreement in terms of impedance progress and lesion depth (4 and 4.5 mm respectively for ex vivo conditions, and approximately 7 and 9 mm respectively for in vivo conditions), although the lesion widths were overestimated by the computer simulations. This could have been due to the method used to assess the thermal lesions; the experimental lesions were assessed by the white coagulation zone, whereas the tissue damage function was used to assess the computational lesions. Conclusions: The experimental results suggest that this applicator could create in vivo lesions to a depth of around 7mm. It was also observed that the thermal lesion is mainly confined to the area between both electrodes, which would allow lesion width to be controlled by selecting a specific applicator design. The comparison between the experimental and computational results suggests that the theoretical model could be usefully applied in further studies of the performance of this device. © 2012 Informa UK Ltd All rights reserved.This work received financial support from the Spanish Plan Nacional de I+D+I del Ministerio de Ciencia e Innovacion TEC2011-27133-C02-(01 and 02), from Universitat Politecnica de Valencia (INNOVA11-01-5502; and PAID-06-11 Ref. 1988). A. Gonzalez-Suarez is the recipient of grant VaLi+D (ACIF/2011/194) from the Generalitat Valenciana. The proof-reading of this paper was funded by the Universitat Politecnica de Valencia, Spain. The authors alone are responsible for the content and writing of the paper.González Suárez, A.; Trujillo Guillen, M.; Burdío Pinilla, F.; Andaluz Martínez, AM.; Berjano Zanón, E. (2012). Feasibility study of an internally cooled bipolar applicator for RF coagulation of hepatic tissue: Experimental and computational study. International Journal of Hyperthermia. 28(7):663-673. https://doi.org/10.3109/02656736.2012.716900S663673287Topp, S. A., McClurken, M., Lipson, D., Upadhya, G. A., Ritter, J. H., Linehan, D., & Strasberg, S. M. (2004). Saline-Linked Surface Radiofrequency Ablation. Annals of Surgery, 239(4), 518-527. doi:10.1097/01.sla.0000118927.83650.a4Gnerlich, J. L., Ritter, J. H., Linehan, D. C., Hawkins, W. G., & Strasberg, S. M. (2009). Saline-Linked Surface Radiofrequency Ablation. Annals of Surgery, 250(1), 96-102. doi:10.1097/sla.0b013e3181ae91afSakamoto, Y., Yamamoto, J., Kokudo, N., Seki, M., Kosuge, T., Yamaguchi, T., … Makuuchi, M. (2004). Bloodless liver resection using the Monopolar Floating Ball plus Ligasure diathermy: Preliminary results of 16 liver resections. World Journal of Surgery, 28(2), 166-172. doi:10.1007/s00268-003-7167-5Poon, R. T., Fan, S. T., & Wong, J. (2005). Liver resection using a saline-linked radiofrequency dissecting sealer for transection of the liver. Journal of the American College of Surgeons, 200(2), 308-313. doi:10.1016/j.jamcollsurg.2004.10.008EVERETT IV, T. H., LEE, K. W., WILSON, E. E., GUERRA, J. M., VAROSY, P. D., & OLGIN, J. E. (2008). Safety Profiles and Lesion Size of Different Radiofrequency Ablation Technologies: A Comparison of Large Tip, Open and Closed Irrigation Catheters. Journal of Cardiovascular Electrophysiology, 20(3), 325-335. doi:10.1111/j.1540-8167.2008.01305.xYokoyama, K., Nakagawa, H., Wittkampf, F. H. M., Pitha, J. V., Lazzara, R., & Jackman, W. M. (2006). Comparison of Electrode Cooling Between Internal and Open Irrigation in Radiofrequency Ablation Lesion Depth and Incidence of Thrombus and Steam Pop. Circulation, 113(1), 11-19. doi:10.1161/circulationaha.105.540062Demazumder, D., Mirotznik, M. S., & Schwartzman, D. (2001). Journal of Interventional Cardiac Electrophysiology, 5(4), 391-400. doi:10.1023/a:1013241927388Cooper, J. M., Sapp, J. L., Tedrow, U., Pellegrini, C. P., Robinson, D., Epstein, L. M., & Stevenson, W. G. (2004). Ablation with an internally irrigated radiofrequency catheter: Learning how to avoid steam pops. Heart Rhythm, 1(3), 329-333. doi:10.1016/j.hrthm.2004.04.019Burdío, F., Grande, L., Berjano, E., Martinez-Serrano, M., Poves, I., Burdío, J. M., … Güemes, A. (2010). A new single-instrument technique for parenchyma division and hemostasis in liver resection: a clinical feasibility study. The American Journal of Surgery, 200(6), e75-e80. doi:10.1016/j.amjsurg.2010.02.020Ríos, J. S., Zalabardo, J. M. S., Burdio, F., Berjano, E., Moros, M., Gonzalez, A., … Güemes, A. (2011). Single Instrument for Hemostatic Control in Laparoscopic Partial Nephrectomy in a Porcine Model Without Renal Vascular Clamping. Journal of Endourology, 25(6), 1005-1011. doi:10.1089/end.2010.0557Dorcaratto, D., Burdío, F., Fondevila, D., Andaluz, A., Poves, I., Martinez, M. A., … Grande, L. (2012). Laparoscopic Distal Pancreatectomy: Feasibility Study of Radiofrequency-Assisted Transection in a Porcine Model. Journal of Laparoendoscopic & Advanced Surgical Techniques, 22(3), 242-248. doi:10.1089/lap.2011.0417Zeh, A., Messer, J., Davis, J., Vasarhelyi, A., & Wohlrab, D. (2010). The Aquamantys System—An Alternative To Reduce Blood Loss in Primary Total Hip Arthroplasty? The Journal of Arthroplasty, 25(7), 1072-1077. doi:10.1016/j.arth.2009.10.008Sprunger, J., & Herrell, S. D. (2005). Partial Laparoscopic Nephrectomy Using Monopolar Saline-Coupled Radiofrequency Device: Animal Model and Tissue Effect Characterization. Journal of Endourology, 19(4), 513-519. doi:10.1089/end.2005.19.513Voeller, R. K., Zierer, A., Lall, S. C., Sakamoto, S., Schuessler, R. B., & Damiano, R. J. (2010). Efficacy of a novel bipolar radiofrequency ablation device on the beating heart for atrial fibrillation ablation: A long-term porcine study. The Journal of Thoracic and Cardiovascular Surgery, 140(1), 203-208. doi:10.1016/j.jtcvs.2009.06.034Pai, M., Spalding, D., Jiao, L., & Habib, N. (2012). Use of Bipolar Radiofrequency in Parenchymal Transection of the Liver, Pancreas and Kidney. Digestive Surgery, 29(1), 43-47. doi:10.1159/000335732Berjano, E. J. (2006). BioMedical Engineering OnLine, 5(1), 24. doi:10.1186/1475-925x-5-24Tungjitkusolmun, S., Staelin, S. T., Haemmerich, D., Jang-Zern Tsai, Hong Cao, Webster, J. G., … Vorperian, V. R. (2002). Three-dimensional finite-element analyses for radio-frequency hepatic tumor ablation. IEEE Transactions on Biomedical Engineering, 49(1), 3-9. doi:10.1109/10.972834Doss, J. D. (1982). Calculation of electric fields in conductive media. Medical Physics, 9(4), 566-573. doi:10.1118/1.595107Abraham, J. P., & Sparrow, E. M. (2007). A thermal-ablation bioheat model including liquid-to-vapor phase change, pressure- and necrosis-dependent perfusion, and moisture-dependent properties. International Journal of Heat and Mass Transfer, 50(13-14), 2537-2544. doi:10.1016/j.ijheatmasstransfer.2006.11.045Jo, B., & Aksan, A. (2010). Prediction of the extent of thermal damage in the cornea during conductive keratoplasty. Journal of Thermal Biology, 35(4), 167-174. doi:10.1016/j.jtherbio.2010.02.004Pearce, J., Panescu, D., & Thomsen, S. (2005). Simulation of diopter changes in radio frequency conductive keratoplasty in the cornea. Modelling in Medicine and Biology VI. doi:10.2495/bio050451Yang, D., Converse, M. C., Mahvi, D. M., & Webster, J. G. (2007). Expanding the Bioheat Equation to Include Tissue Internal Water Evaporation During Heating. IEEE Transactions on Biomedical Engineering, 54(8), 1382-1388. doi:10.1109/tbme.2007.890740Zhao, G., Zhang, H.-F., Guo, X.-J., Luo, D.-W., & Gao, D.-Y. (2007). Effect of blood flow and metabolism on multidimensional heat transfer during cryosurgery. Medical Engineering & Physics, 29(2), 205-215. doi:10.1016/j.medengphy.2006.03.005Pätz T, Körger T, Preusser T, Simulation of radiofrequency ablation including water evaporation. In: IFMBE Proceedings of the World Congress on Medical Physics and Biomedical Engineering 25/IV, 2009, 1287–1290Berjano, E. J., Burdío, F., Navarro, A. C., Burdío, J. M., Güemes, A., Aldana, O., … Gregorio, M. A. de. (2006). Improved perfusion system for bipolar radiofrequency ablation of liver: preliminary findings from a computer modeling study. Physiological Measurement, 27(10), N55-N66. doi:10.1088/0967-3334/27/10/n03Burdío, F., Berjano, E. J., Navarro, A., Burdío, J. M., Grande, L., Gonzalez, A., … Lequerica, J. L. (2009). Research and development of a new RF-assisted device for bloodless rapid transection of the liver: Computational modeling and in vivo experiments. BioMedical Engineering OnLine, 8(1), 6. doi:10.1186/1475-925x-8-6Chang, I. A., & Nguyen, U. D. (2004). BioMedical Engineering OnLine, 3(1), 27. doi:10.1186/1475-925x-3-27Beop-Min Kim, Jacques, S. L., Rastegar, S., Thomsen, S., & Motamedi, M. (1996). Nonlinear finite-element analysis of the role of dynamic changes in blood perfusion and optical properties in laser coagulation of tissue. IEEE Journal of Selected Topics in Quantum Electronics, 2(4), 922-933. doi:10.1109/2944.577317Panescu, D., Whayne, J. G., Fleischman, S. D., Mirotznik, M. S., Swanson, D. K., & Webster, J. G. (1995). Three-dimensional finite element analysis of current density and temperature distributions during radio-frequency ablation. IEEE Transactions on Biomedical Engineering, 42(9), 879-890. doi:10.1109/10.412649González-Suárez A, Alba J, Trujillo M, Berjano E, Experimental and theoretical study of an internally cooled bipolar electrode for RF coagulation of biological tissues. Conf Proc IEEE Eng Med Biol Soc 2011;6878–6881Rosenberg, A. G. (2007). Reducing Blood Loss in Total Joint Surgery With a Saline-Coupled Bipolar Sealing Technology. The Journal of Arthroplasty, 22(4), 82-85. doi:10.1016/j.arth.2007.02.018PETERSEN, H. H., ROMAN-GONZALEZ, J., JOHNSON, S. B., HASTRUP SVENDSEN, J., HAUNSO, S., & PACKER, D. L. (2004). Mechanisms for Enlarging Lesion Size During Irrigated Tip Radiofrequency Ablation:. Is There a Virtual Electrode Effect? Journal of Interventional Cardiology, 17(3), 171-177. doi:10.1111/j.1540-8183.2004.09879.

HOXB13 is downregulated in colorectal cancer to confer TCF4-mediated transactivation

Mutations in the Wnt signalling cascade are believed to cause aberrant proliferation of colorectal cells through T-cell factor-4 (TCF4) and its downstream growth-modulating factors. HOXB13 is exclusively expressed in prostate and colorectum. In prostate cancers, HOXB13 negatively regulates β-catenin/TCF4-mediated transactivation and subsequently inhibits cell growth. To study the role of HOXB13 in colorectal tumorigenesis, we evaluated the expression of HOXB13 in 53 colorectal tumours originated from the distal left colon to rectum with their matching normal tissues using quantitative RT–PCR analysis. Expression of HOXB13 is either lost or diminished in 26 out of 42 valid tumours (62%), while expression of TCF4 RNA is not correlated with HOXB13 expression. TCF4 promoter analysis showed that HOXB13 does not regulate TCF4 at the transcriptional level. However, HOXB13 downregulated the expression of TCF4 and its target gene, c-myc, at the protein level and consequently inhibited β-catenin/TCF-mediated signalling. Functionally, forced expression of HOXB13 drove colorectal cancer (CRC) cells into growth suppression. This is the first description of the downregulation of HOXB13 in CRC and its mechanism of action is mediated through the regulation of TCF4 protein stability. Our results suggest that loss of HOXB13 may be an important event for colorectal cell transformation, considering that over 90% of colorectal tumours retain mutations in the APC/β-catenin pathway

BRAF Activation Initiates but Does Not Maintain Invasive Prostate Adenocarcinoma

Prostate cancer is the second leading cause of cancer-related deaths in men. Activation of MAP kinase signaling pathway has been implicated in advanced and androgen-independent prostate cancers, although formal genetic proof has been lacking. In the course of modeling malignant melanoma in a tyrosinase promoter transgenic system, we developed a genetically-engineered mouse (GEM) model of invasive prostate cancers, whereby an activating mutation of BRAFV600E–a mutation found in ∼10% of human prostate tumors–was targeted to the epithelial compartment of the prostate gland on the background of Ink4a/Arf deficiency. These GEM mice developed prostate gland hyperplasia with progression to rapidly growing invasive adenocarcinoma without evidence of AKT activation, providing genetic proof that activation of MAP kinase signaling is sufficient to drive prostate tumorigenesis. Importantly, genetic extinction of BRAFV600E in established prostate tumors did not lead to tumor regression, indicating that while sufficient to initiate development of invasive prostate adenocarcinoma, BRAFV600E is not required for its maintenance

An investigation of WNT pathway activation and association with survival in central nervous system primitive neuroectodermal tumours (CNS PNET)

Central nervous system primitive neuroectodermal tumours (CNS PNET) are high-grade, predominantly paediatric, brain tumours. Previously they have been grouped with medulloblastomas owing to their histological similarities. The WNT/β-catenin pathway has been implicated in many tumour types, including medulloblastoma. On pathway activation β-catenin (CTNNB1) translocates to the nucleus, where it induces transcription of target genes. It is commonly upregulated in tumours by mutations in the key pathway components APC and CTNNB1. WNT/β-catenin pathway status was investigated by immunohistochemical analysis of CTNNB1 and the pathway target cyclin D1 (CCND1) in 49 CNS PNETs and 46 medulloblastomas. The mutational status of APC and CTNNB1 (β-catenin) was investigated in 33 CNS PNETs and 22 medulloblastomas. CTNNB1 nuclear localisation was seen in 36% of CNS PNETs and 27% of medulloblastomas. A significant correlation was found between CTNNB1 nuclear localisation and CCND1 levels. Mutations in CTNNB1 were identified in 4% of CNS PNETs and 20% of medulloblastomas. No mutations were identified in APC. A potential link between the level of nuclear staining and a better prognosis was identified in the CNS PNETs, suggesting that the extent of pathway activation is linked to outcome. The results suggest that the WNT/β-catenin pathway plays an important role in the pathogenesis of CNS PNETs. However, activation is not caused by mutations in CTNNB1 or APC in the majority of CNS PNET cases

Secreted Frizzled-related protein-1 is a negative regulator of androgen receptor activity in prostate cancer

Secreted Frizzled-related protein-1 (sFRP1) associates with Wnt proteins and its loss can lead to activation of Wnt/β-catenin signalling. It is frequently downregulated in cancer, including prostate cancer, but its function in prostate cancer is unclear because it can increase proliferation of prostate epithelial cells. We investigated the function of sFRP1 in androgen-dependent prostate cancer and found that sFRP1 inhibited androgen receptor (AR) transcriptional activity. In addition, sFRP1 inhibited the proliferation of androgen-dependent LNCaP cells but not of an androgen-independent subline LNCaP-r, suggesting a role in androgen-dependent growth. The inhibition of AR by sFRP1 was unaffected by co-expression of Wnt3a, stabilised β-catenin or β-catenin shRNA, suggesting it does not involve Wnt/β-catenin signalling. Wnt5a also inhibited AR and expression of Wnt5a and sFRP1 together did not further inhibit AR, suggesting that Wnt5a and sFRP1 activate the same signal(s) to inhibit AR. However, sFRP1 inhibition of AR was unaffected by inhibitors of kinases involved in Wnt/Ca2+ and Wnt/planar cell polarity non-canonical Wnt signalling. Interestingly, the cysteine-rich domain of sFRP1 interacted with Frizzled receptors expressed in prostate cancer cells, suggesting that sFRP1/Frizzled complexes activate a signal that leads to repression of AR. Taken together, these observations highlight the function of β-catenin-independent Wnt signalling in the control of AR activity and provide one explanation for sFRP1 downregulation in prostate cancer

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.