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NASA NTRS2014

Towards a Biosynthetic UAV

We are currently working on a series of projects towards the construction of a fully biological unmanned aerial vehicle (UAV) for use in scientific and humanitarian missions. The prospect of a biologically-produced UAV presents numerous advantages over the current manufacturing paradigm. First, a foundational architecture built by cells allows for construction or repair in locations where it would be difficult to bring traditional tools of production. Second, a major limitation of current research with UAVs is the size and high power consumption of analytical instruments, which require bulky electrical components and large fuselages to support their weight. By moving these functions into cells with biosensing capabilities - for example, a series of cells engineered to report GFP, green fluorescent protein, when conditions exceed a certain threshold concentration of a compound of interest, enabling their detection post-flight - these problems of scale can be avoided. To this end, we are working to engineer cells to synthesize cellulose acetate as a novel bioplastic, characterize biological methods of waterproofing the material, and program this material's systemic biodegradation. In addition, we aim to use an "amberless" system to prevent horizontal gene transfer from live cells on the material to microorganisms in the flight environment.

Biological↗

NASA NTRS1999

GH/IGF-I Transgene Expression on Muscle Homeostasis

We propose to test the hypothesis that the growth hormone/ insulin like growth factor-I axis through autocrine/paracrine mechanisms may provide long term muscle homeostasis under conditions of prolonged weightlessness. As a key alternative to hormone replacement therapy, ectopic production of hGH, growth hormone releasing hormone (GHRH), and IGF-I will be studied for its potential on muscle mass impact in transgenic mice under simulated microgravity. Expression of either hGH or IGF-I would provide a chronic source of a growth-promoting protein whose biosynthesis or secretion is shut down in space. Muscle expression of the IGF-I transgene has demonstrated about a 20% increase in hind limb muscle mass over control nontransgenic litter mates. These recent experiments, also establish the utility of hind-limb suspension in mice as a workable model to study atrophy in weight bearing muscles. Thus, transgenic mice will be used in hind-limb suspension models to determine the role of GH/IGF-I on maintenance of muscle mass and whether concentric exercises might act in synergy with hormone treatment. As a means to engineer and ensure long-term protein production that would be workable in humans, gene therapy technology will be used by to monitor muscle mass preservation during hind-limb suspension, after direct intramuscular injection of a genetically engineered muscle-specific vector expressing GHRH. Effects of this gene-based therapy will be assessed in both fast twitch (medial gastrocnemius) and slow twitch muscle (soleus). End-points include muscle size, ultrastructure, fiber type, and contractile function, in normal animals, hind limb suspension, and reambutation.

Schwartz, Robert J.↗

NASA NTRSDate not supplied

3D Construction of Biologically Derived Materials

System for the 3D Construction of Biologically Derived Materials, Structures, and Parts NASA has developed a novel approach for macroscale biomaterial production by combining synthetic biology with 3D printing. Cells are biologically engineered to deposit desired materials, such as proteins or metals, derived from locally available resources. The bioengineered cells build different materials in a specified 3D pattern to produce novel microstructures with precise molecular composition, thickness, print pattern, and shape. Scaffolds and reagents can be used for further control over material product. This innovation provides modern design and fabrication techniques for custom-designed organic or organic-inorganic composite biomaterials produced from limited resources. Benefits Conserves resources. Few raw or bulk starting materials needed Enables custom design of diverse materials Fast, portable, macroscale, on-demand manufacturing High-fidelity microstructures Uses commercially available parts Applications Biomaterials, biotechnology Organic-inorganic composite materials On-demand manufacturing In situ resource utilization Space stations Military Infrastructure materials The Technology Once genes for a desired material type, delivery mode, control method and affinity have been chosen, assembling the genetic components and creating the cell lines can be done with well-established synthetic biology techniques. A 3D microdeposition system is used to make a 3D array of these cells in a precise, microstructure pattern and shape. The engineered cells are suspended in a printable 'ink'. The 3D microdeposition system deposits minute droplets of the cells onto a substrates surface in a designed print pattern. Additional printer passes thicken the material. The cell array is fed nutrients and reagents to activate the engineered genes within the cells to create and deposit the desired molecules. These molecules form the designed new material. If desired, the cells may be removed by flushing. The end product is thus a 3D composite microstructure comprising the novel material. This innovation provides a fast, controlled production of natural, synthetic, and novel biomaterials with minimum resource overhead and reduced pre- and post-processing requirements.

3D↗

NASA NTRS1999

Tissue Engineered Skeletal Myofibers can Directly "Sense" Gravitational Force Changes

Long-term manned space flight requires a better understanding of skeletal muscle atrophy resulting from microgravity. Atrophy most likely results from changes at both the systemic level (e.g. decreased circulating growth hormone, increased circulating glucocorticoids) and locally (e.g. decreased myofiber resting tension). Differentiated skeletal myofibers in tissue culture have provided a model system over the last decade for gaining a better understanding of the interactions of exogenous growth factors, endogenous growth factors, and muscle fiber tension in regulating protein turnover rates and muscle cell growth. Tissue engineering these cells into three dimensional bioartificial muscle (BAM) constructs has allowed us to extend their use to Space flight studies for the potential future development of countermeasures. Embryonic avian muscle cells were isolated and BAMs tissue engineered as described previously. The myoblasts proliferate and fuse into aligned postmitotic myofibers after ten to fourteen days in vitro. A cylindrical muscle-like structure containing several thousand myofibers is formed which is approximately 30 mm in length, 2-3 mm in diameter, and attached at each end. For the Space Shuttle experiments, the BAMs were transferred to 55 mL bioreactor cartridges (6 BAMs/cartridge). At Kennedy Space Center, the cartridges were mounted in two Space Tissue Loss (STL) Modules (three to four cartridges per Module) and either maintained as ground controls or loaded in a Mid-Deck locker of the Space Shuttle. The BAM cartridges were continuously perfused during the experiment at 1.5 mL/ min with tissue culture medium. Eighteen BAMs were flown for nine days on Mission STS66 while eighteen BAMs served as ground controls. The complete experiment was repeated on Mission STS77 with twenty four BAMs in each group. BAMs could be maintained in a healthy state for at least 30 days in the perfusion bioreactor cartridges. The BAM muscle fibers directly detected both the loss of gravity and the reloading effects of 1 x g. While total cellular metabolism and total protein degradation rates were not altered during 9 to 10 days in Space, protein synthesis rates were significantly reduced and resulted in significant myofiber atrophy compared to ground controls. One g reloading of the flight muscle cells post-flight significantly increased protein synthesis rates and the synthesis rates of myosin heavy chain, fibronectin, and collagen. Tissue cultured muscle cells can directly "sense" changes in gravity and provide a valid model to begin the study of countermeasures. Based on our ground based experiments, and the experiments of others, growth hormone and/or insulin-like growth factors are attractive protein therapeutics which may assisting attenuating skeletal muscle wasting in space. Our laboratory is developing a new cell-based delivery system for this and other potential therapeutic factors for attenuating muscle and bone wasting.

Vandenburgh, Herman H.↗

NASA NTRS1999

Tissue Engineering Organs for Space Biology Research

Vandenburgh, H. H.↗

NASA NTRS2002

Tissue engineering skeletal muscle for orthopaedic applications

With current technology, tissue-engineered skeletal muscle analogues (bioartificial muscles) generate too little active force to be clinically useful in orthopaedic applications. They have been engineered genetically with numerous transgenes (growth hormone, insulinlike growth factor-1, erythropoietin, vascular endothelial growth factor), and have been shown to deliver these therapeutic proteins either locally or systemically for months in vivo. Bone morphogenetic proteins belonging to the transforming growth factor-beta superfamily are osteoinductive molecules that drive the differentiation pathway of mesenchymal cells toward the chondroblastic or osteoblastic lineage, and stimulate bone formation in vivo. To determine whether skeletal muscle cells endogenously expressing bone morphogenetic proteins might serve as a vehicle for systemic bone morphogenetic protein delivery in vivo, proliferating skeletal myoblasts (C2C12) were transduced with a replication defective retrovirus containing the gene for recombinant human bone morphogenetic protein-6 (C2BMP-6). The C2BMP-6 cells constitutively expressed recombinant human bone morphogenetic protein-6 and synthesized bioactive recombinant human bone morphogenetic protein-6, based on increased alkaline phosphatase activity in coincubated mesenchymal cells. C2BMP-6 cells did not secrete soluble, bioactive recombinant human bone morphogenetic protein-6, but retained the bioactivity in the cell layer. Therefore, genetically-engineered skeletal muscle cells might serve as a platform for long-term delivery of osteoinductive bone morphogenetic proteins locally.

Review↗

NASA NTRS2007

Recombinant Collagenlike Proteins

A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

Fertala, Andzej↗

NASA NTRS2005

The Future of Bio-technology

Hosts of technologies, most notably in electronics, have been on the path of miniaturization for decades and in 2005 they have crossed the threshold of the nano-scale. Crossing the nano-scale threshold is a milestone in miniaturization, setting impressive new standards for component-packing densities. It also brings technology to a scale at which quantum effects and fault tolerance play significant roles and approaches the feasible physical limit form many conventional "top-down" manufacturing methods. I will suggest that the most formidable manufacturing problems in nanotechnology will be overcome and major breakthroughs will occur in a host of technologies, when nanotechnology converges with bio-technology; i.e. I will argue that the future of bio-technology is in nanotechnology. In 2005, methods in molecular biology, microscopy, bioinformatics, biochemistry, and genetic engineering have focused considerable attention on the nano-scale. On this scale, biology is a kind of recursive chemistry in which molecular recognition, self-assembly, self-organization and self-referencing context-control lead to the emergence of the complexity of structures and processes that are fundamental to all life forms. While we are still far from understanding this complexity, we are on the threshold of being able to use at least some of these biological properties for .technology. I will discuss the use of biomolecules, such as DNA, RNA, and proteins as "tools" for the bio-technologist of the future. More specifically, I will present in some detail an example of how we are using a genetically engineered 60-kDa protein (HSP60) from an organism living in near boiling sulfuric acid to build nano-scale templates for arranging metallic nanoparticles. These "extremophile" HSP60s self-assemble into robust double-ring structures called "chaperonins," which further assemble into filaments and arrays with nanometer accuracy. I will discuss our efforts to use chaperonins to organize quantum dots, electronic and magnetic nano-particles for electronic and photonic applications.

Trent, Jonathan↗

NASA NTRS2002

Peptides at Membrane Surfaces and their Role in Prebiotic Evolution

Protocells had to transport ions and organic matter across membranes separating the interior of the cell from the environment, capture and utilize energy and transduce environmental signals. In a series of detailed, molecular-level computer simulations we show how these peptides in contact with membranes can acquire ordered structures and functions. We have investigated the stability of a simple alpha-helical peptide containing Leucine (L) and Serine (S) of the form (LSLLLSL)3 in a model membrane system. The parallel in-plane state is the most stable configuration. The transmembrane state is metastable, and about 15 kcal/mol is required to insert the peptide into the membrane. We investigated dimes of both (LSLLLSL)3 and glycophorin A, and show how the free energy of helix association can, at least partially, offset the free energy of insertion. We have also investigated the transmembrane pore of the influenza M2 protein. This aggregate of four identical alpha-helices, each built of 25 amino acids, forms an efficient and selective voltage-gated proton channel. Our simulations explain the gating mechanism, which can involve strands of hydrogen-bonded water through the pore or proton transfer through tautomerization of protein residues. The channel can be re-engineered to act as a simple proton pump.

Pohorille, Andrew↗

NASA NTRS2001

Shear stress reduces protease activated receptor-1 expression in human endothelial cells

Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

Non-NASA Center↗

NASA NTRS2003

Using Proteins to Build Nanocomputers

How low can we go? This is the question challenging scientists who want to build ever-smaller electronic components. As famed physicist Richard Feynmann said in 1959, 'there is plenty of room at the bottom,' meaning at the atomic or nanometric scale. But even Feynmann could only guess at how we might eventually build things 'down there.' As current lithographic techniques for etching semiconductor circuits rapidly approach their physical limits, scientists are looking for new and less expensive ways to fabricate circuits at infinitesimally small scales. NASA scientists have discovered a new tool that may help accomplish this goal - proteins. The basic building blocks of life may be harnessed to build computers and sensors on the nanoscale. Proteins provide self-assembling structures that can be engineered to build ordered arrays of quantum dots.

Laufenberg, Larry↗

NASA NTRS1995

Immunohistochemical detection of active transforming growth factor-beta in situ using engineered tissue

The biological activity of transforming growth factor-beta 1 (TGF-beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-beta could provide insight into TGF-beta activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-beta or constitutively active TGF-beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-beta activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-beta versus latent TGF-beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a protein.

NASA Discipline Radiation Health↗

NASA NTRS2019

Employing Automated Experimental Evolution to Understand Survival Strategies of Lab-Grown Extremophiles

Experimental evolution (EE) exposes microbes to intentional stressors to improve resistance through artificial mutation. The resulting changes to metabolic pathways, protein structure, and genetic sequences, along with traditional genetic engineering tools, to can help understand the mechanisms of improved tolerance. An automated experimental set-up -- the Automated Adaptive Directed Evolution Chamber (AADEC) -- with minimal scope for human interference was developed at NASA Ames. A second- generation device integrating more real-time biochemical sensors has been developed recently. Added sensors include pH for indicating metabolic products, oxidation-reduction potential (ORP) for indicating available/consumed metabolic energy, dissolved oxygen (DO) for indicating aerobic/anaerobic growth cycles, and electrical conductivity (EC) as an additional indicator of metabolic products. With four additional sensors, the system is biochemically more informative in real-time. More importantly, each sensor parameter can be used as a selection pressure, individually or in combination with others, to artificially create and control inhospitable environments analogous to extremophile habitats for microbial growth in the lab. Potential stressors to be added in the future include thermal, reactive oxygen species, metal-ion concentrations, and varying nutrient availability.

Automated↗

NASA NTRS1992

More Genetic Engineering With Cloned Hemoglobin Genes

Cells modified to enhance growth and production of proteins. Method for enhancing both growth of micro-organisms in vitro and production of various proteins or metalbolites in these micro-organisms provides for incorporation of selected chromosomal or extrachormosomal deoxyribonucleic acid (DNA) sequences into micro-organisms from other cells or from artificial sources. Incorporated DNA includes parts encoding desired product(s) or characteristic(s) of cells and parts that control expression of productor characteristic-encoding parts in response to variations in environment. Extended method enables increased research into growth of organisms in oxygen-poor environments. Industrial applications found in enhancement of processing steps requiring oxygen in fermentation, enzymatic degradation, treatment of wastes containing toxic chemicals, brewing, and some oxidative chemical reactions.

Bailey, James E.↗

NASA NTRS2003

Membrane peptides and their role in protobiological evolution

How simple membrane peptides performed such essential protocellular functions as transport of ions and organic matter across membranes separating the interior of the cell from the environment, capture and utilization of energy, and transduction of environmental signals, is a key question in protobiological evolution. On the basis of detailed, molecular-level computer simulations we explain how these peptides fold at water-membrane interfaces, insert into membranes, self-assemble into higher-order structures and acquire functions. We have investigated the interfacial behavior and folding of several peptides built of leucine and glutamine residues and have demonstrated that many of them tend to adopt ordered structures. Further, we have studied the insertion of an alpha-helical peptide containing leucine (L) and serine (S) of the form (LSLLLSL)3 into a model membrane. The transmembrane state is metastable, and approximately 15 kcal mol(-1) is required to insert the peptide into the membrane. Investigations of dimers formed by (LSLLLSL)3 and glycophorin A demonstrate how the favorable free energy of helix association can offset the unfavorable free energy of insertion, leading to self-assembly of peptide helices in the membrane. An example of a self-assembled structure is the tetrameric transmembrane pore of the influenza virus M2 protein, which is an efficient and selective voltage-gated proton channel. Our simulations explain the gating mechanism and provide guidelines how to re-engineer the channel to act as a simple proton pump. In general, emergence of integral membrane proteins appears to be quite feasible and may be easier to envision than the emergence of water-soluble proteins.

Models, Chemical↗

NASA NTRS2014

BioSentinel: Monitoring DNA Damage Repair Beyond Low Earth Orbit on a 6U Nanosatellite

We are designing and developing a 6U nanosatellite as a secondary payload to fly aboard NASAs Space Launch System (SLS) Exploration Mission (EM) 1, scheduled for launch in late 2017. For the first time in over forty years, direct experimental data from biological studies beyond low Earth orbit (LEO) will be obtained during BioSentinels 12- to 18-month mission. BioSentinel will measure the damage and repair of DNA in a biological organism and allow us to compare that to information from onboard physical radiation sensors. This data will be available for validation of existing models and for extrapolation to humans.The BioSentinel experiment will use the organism Saccharomyces cerevisiae (yeast) to report DNA double-strand-break (DSB) events that result from space radiation. DSB repair exhibits striking conservation of repair proteins from yeast to humans. The flight strain will include engineered genetic defects that prevent growth and division until a radiation-induced DSB activates the yeasts DNA repair mechanisms. The triggered culture growth and metabolic activity directly indicate a DSB and its repair. The yeast will be carried in the dry state in independent microwells with support electronics. The measurement subsystem will sequentially activate and monitor wells, optically tracking cell growth and metabolism. BioSentinel will also include TimePix radiation sensors implemented by JSCs RadWorks group. Dose and Linear Energy Transfer (LET) data will be compared directly to the rate of DSB-and-repair events measured by the S. cerevisiae biosentinels. BioSentinel will mature nanosatellite technologies to include: deep space communications and navigation, autonomous attitude control and momentum management, and micropropulsion systems to provide an adaptable nanosatellite platform for deep space uses.

cosmic rays↗

NASA NTRS1982

Isoelectric focusing in space

The potential of space electrophoresis is conditioned by the fact that all electrophoretic techniques require the suppression of gravity-caused convection. Isoelectric focusing (IEF) is a powerful variant of electrophoresis, in which amphoteric substances are separated in a pH gradient according to their isoelectric points. A new apparatus for large scale IEF, utilizing a recycling principle, has been developed. In the ground-based prototype, laminar flow is provided by a series of parallel filter elements. The operation of the apparatus is monitored by an automated array of pH and ultraviolet absorption sensors under control of a desk-top computer. The apparatus has proven to be useful for the purification of a variety of enzymes, snake venom proteins, peptide hormones, and other biologicals, including interferon produced by genetic engineering techniques. In planning for a possible space apparatus, a crucial question regarding electroosmosis needs to be addressed To solve this problem, simple focusing test modules are planned for inclusion in an early Shuttle flight.

Bier, M.↗

NASA NTRS2012

Spinoff 2011

Topics include: Bioreactors Drive Advances in Tissue Engineering; Tooling Techniques Enhance Medical Imaging; Ventilator Technologies Sustain Critically Injured Patients; Protein Innovations Advance Drug Treatments, Skin Care; Mass Analyzers Facilitate Research on Addiction; Frameworks Coordinate Scientific Data Management; Cameras Improve Navigation for Pilots, Drivers; Integrated Design Tools Reduce Risk, Cost; Advisory Systems Save Time, Fuel for Airlines; Modeling Programs Increase Aircraft Design Safety; Fly-by-Wire Systems Enable Safer, More Efficient Flight; Modified Fittings Enhance Industrial Safety; Simulation Tools Model Icing for Aircraft Design; Information Systems Coordinate Emergency Management; Imaging Systems Provide Maps for U.S. Soldiers; High-Pressure Systems Suppress Fires in Seconds; Alloy-Enhanced Fans Maintain Fresh Air in Tunnels; Control Algorithms Charge Batteries Faster; Software Programs Derive Measurements from Photographs; Retrofits Convert Gas Vehicles into Hybrids; NASA Missions Inspire Online Video Games; Monitors Track Vital Signs for Fitness and Safety; Thermal Components Boost Performance of HVAC Systems; World Wind Tools Reveal Environmental Change; Analyzers Measure Greenhouse Gasses, Airborne Pollutants; Remediation Technologies Eliminate Contaminants; Receivers Gather Data for Climate, Weather Prediction; Coating Processes Boost Performance of Solar Cells; Analyzers Provide Water Security in Space and on Earth; Catalyst Substrates Remove Contaminants, Produce Fuel; Rocket Engine Innovations Advance Clean Energy; Technologies Render Views of Earth for Virtual Navigation; Content Platforms Meet Data Storage, Retrieval Needs; Tools Ensure Reliability of Critical Software; Electronic Handbooks Simplify Process Management; Software Innovations Speed Scientific Computing; Controller Chips Preserve Microprocessor Function; Nanotube Production Devices Expand Research Capabilities; Custom Machines Advance Composite Manufacturing; Polyimide Foams Offer Superior Insulation; Beam Steering Devices Reduce Payload Weight; Models Support Energy-Saving Microwave Technologies; Materials Advance Chemical Propulsion Technology; and High-Temperature Coatings Offer Energy Savings.

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