The identification of novel genes involved in copper and oxygen metabolism

Copper (Cu) is an essential trace dietary metal which is also potentially toxic. Cells have evolved an intricate system of transporters and chaperones to carefully regulate Cu homeostasis. Cu uptake is mediated by the Cu1+-specific permease, CTR1. Cytoplasmic Cu is delivered to specific compartments via a series of metallochaperones. In the trans-Golgi network (TGN), the ATP7A or ATP7B proteins are required to pump Cu into the secretory pathway to various cuproenzymes. ATP7A also plays a protective role against Cu toxicity by trafficking to the plasma membrane to export Cu from the cell. Metallothioneins (MTs) are another type of protein that plays a protective role against Cu toxicity by sequestering excess Cu in the cytoplasm. There are four isoforms of MT in mice of which MT I and MT II are ubiquitously expressed. Our preliminary data demonstrates the essentiality of ATP7A, MTI and MTII in copper tolerance. We generated a fibroblast cell line in which Atp7a, Mt-I, and Mt-II genes were deleted (Atp7a-/Mt-), which resulted in sensitivity of these cells to sub-micromolar levels of copper. Using a genome-wide CRISPR-Cas9 deletion screen, we identified a novel gene, VHL (von Hippel-Lindau), that when deleted confers Cu tolerance to Atp7a-/Mt- cells. The VHL protein is a ubiquitin-ligase responsible for the degradation of HIF transcription factors, which are the major regulators of gene expression in response to hypoxia. We show that VHL deletion confers Cu tolerance by stimulating the expression of the ATP7B copper transporter. Pharmacological inhibition of VHL-HIF1[alpha] by Roxadustat (a prolyl hydroxylase inhibitor), which is clinically used to treat anemia, conferred resistance to copper by upregulating ATP7B. Importantly, Roxadustat was found to increase hepatic ATP7B expression in vivo. These novel data identify a previously unknown link between copper homeostasis and oxygen sensing and have potential translational implications for the treatment of Wilson disease, a copper overload condition cause by insufficient expression of ATP7B.Includes bibliographical references (pages 102-118)

Small molecule inhibitors of ATP7A as novel theraphies for cancer

Recent studies suggest that trace element copper (Cu) plays a key role in cancer progression. Cu dependent signaling pathways in cancer cells are poorly understood and are being actively investigated. In a recently published PNAS paper, we demonstrated that ATP7A copper transporter promotes breast and lung tumor growth and spread in mice by activating pro cancerous enzyme, lysyl oxidase (LOX). Therefore, we propose that blocking ATP7A using small molecule inhibitors could be a powerful approach in blocking cancer progression. We screened for inhibitors of ATP7A using a structure based virtual screen of ATP7A interacting drug like compounds, resulting in identification of our lead compound, MKV3. We used microscale thermophoresis and standard enzyme activity assays to measure the binding affinity of MKV3 to ATP7A and test the effect of MKV3 on ATP7A dependent LOX and tyrosinase enzymes. In vitro scratch and tumorigenesis assays were performed to test the effect of MKV3 on cancer cell migration and tumor growth. Microscale thermophoresis experiments revealed that MKV3 binds to ATP7A with nanomolar affinity and is a potent inhibitor of ATP7A dependent LOX activity and cell migration of 4T1 breast, LLC and A549 lung cancer cells. Moreover, MKV3 inhibited ATP7A dependent tyrosinase activity in B16 melanoma cells and suppressed B16 tumorigenesis in vivo. In summary, these studies have identified a novel first in class high affinity inhibitor of ATP7A and provide a framework to design MKV3 derivative s with improved therapeutic efficacy in mouse models of cancer. Our findings have the potential for a sustained and powerful impact in cancer therapy.NIH R 01 fundingShanbhag Vinit, 1,4 ; Gudekar Nikita 2 ; Kamlendra Singh 3 ; Petris, Michael 1,2,4 (1 - Department of Biochemistry, 2 - Genetics Area Program, 3 - Department of Veterinary Pathobiology, 4 - Department of Opthalmology, University of Missouri, Columbia 65211

Design and development of energy efficient continuous cooking system

© 2015 Elsevier Ltd. Abstract For large scale cooking, it is desirable to implement continuous cooking. In addition to the usual advantages of continuous cooking (size of cooker, uniform cooked quality, possibility of process control), there is a possibility of increasing the thermal efficiency by preheating water against the hot cooked food leaving the cooker. In the present work, solid and liquid phase residence time distribution (RTD) have been measured in 120 mm diameter and 1.6 m long continuous cooker. Three levels of screw speed (1, 3 and 5 rpm), two levels of solid flow rate (12and 24 kg/h) and three levels of liquid flow rate (15, 25 and 35 lph) were selected. The solid phase was always found to move in a plug flow manner as the minimum Peclet number was found to be 81.5. This information was found to be useful for deciding the capacity of the continuous cooker. Thus, the time required for batch cooking was found to be equal to the minimum residence time in a continuous cooker at equivalent operating condition of temperature

A novel method to improve the efficiency of a cooking device via thermal insulation

We propose a method of finding the transient temperature variation in an insulated cooking device. We also report a means of optimising the thickness of insulation. The cooking device is a double walled cylindrical vessel with spacing of 5–20 mm between the vertical walls (width) and spacing of 560 or 870 mm between top and bottom surfaces (height). The height to width ratio (H/L) is between 28 and 174 and Rayleigh number (Ra) is between 907 and 2.61 × 105. First, an energy balance for the cooking device is established. A correlation is developed to predict the heat transfer coefficient (HTC) as a function of Ra and H/L. The method developed for finding the transient variation in temperature has been tested on two cooking device volumes: 120 and 700 lit. Using the optimised parameters, a reduction in heat loss of 22% and 30%, respectively, is observed

Inclined solar chimney for power production

The present concept of solar chimney is a tall vertical chimney constructed at the center of a large area, which is the collector. This creates questions about stability and economic viability of the chimney and also demands elaborate engineering techniques for constructing a tall chimney. We suggest geometry of "Inclined Solar Chimney" (ISC), which is constructed along the face of a high rising mountain, on which maximum solar insolation is incident throughout the year. The chimney and the collector get merged here. This makes the structure stable, cost effective and easy for construction. A mathematical model has been developed considering the total energy balance. It predicts the temperature and velocity and kinetic power of the emerging air draft for some chosen values of other parameters. The model also shows the proportion in which absorbed solar energy is divided into different forms, and hence predicts the dependence of kinetic of emerging air draft upon dimensions of the chimney and properties of materials used. Further, it is shown that external winds enhance the kinetic power of the emerging air. Thus ISC can also harness the wind energy, available at the top of the mountain