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image of Unitary Plan Wind Tunnel 9-by 7-foot Supersonic Test ...

Unitary Plan Wind Tunnel 9-by 7-foot Supersonic Test ...

Dec 01, 2014 · The 9-by 7-foot Supersonic Wind Tunnel (9x7 SWT) facility is part of the Unitary Plan Wind Tunnel (UPWT) complex at NASA's Ames Research Center at Moffett Field, California, where generations of commercial and military aircraft and NASA space vehicles, including the space shuttle, have been designed and tested.Closed circuit, single return, variable density, continuous flow wind tunnel with a Mach range of 1.55 to 2.55..
From: www.nasa.gov

The 9-by 7-foot Supersonic Wind Tunnel (9x7 SWT) facility is part of the Unitary Plan Wind Tunnel (UPWT) complex at NASA's Ames Research Center at Moffett Field, California, where generations of commercial and military aircraft and NASA space vehicles, including the space shuttle, have been designed and tested.

The 9x7 SWT is a closed-return, variable-density tunnel with an asymmetric, sliding-block nozzle. It is one of three separate test sections powered by a common drive system. Interchangeability of models among the UPWT test sections allows testing across a wide range of conditions. Airflow is generated by an 11-stage, axial-flow compressor powered by four variable-speed, wound-rotor induction motors.

The 9x7 SWT continues to provide aerodynamic data for NASA's manned spaceflight efforts whose goal it is to create the rockets and spacecraft necessary to take explorers to Earth orbit, the Moon, and eventually, to Mars.

0.65%-scale sonic boom model in 9-by 7-foot Wind Tunnel at NASA Ames. Image credit: NASA Ames Research Center


Adobe pdf logo  ATP 9-by 7-foot Brochure.pdf

Capability

Operating Characteristics

Test Section Dimensions

Test Section Dimensions Plan View AutoCAD Files:

AutoCAD logo  Version 2000i AutoCAD file (.dwg)

Translation files:

Drawing Exchange File logo  Version 2000 Drawing Exchange file (.dxf)

Initial Graphics Exchange Specification File logo  Initial Graphics Exchange Specification file (.igs)
 

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Test Section Dimensions Elevation View AutoCAD Files:

AutoCAD logo  Version 2000i AutoCAD file (.dwg)

Translation files:

Drawing Exchange File logo  Version 2000 Drawing Exchange file (.dxf)

Initial Graphics Exchange Specification File logo  Initial Graphics Exchange Specification file (.igs)

*Note: Right Click (PC) / Control Click (Mac) and "Save As..." to Download Files. To Open, use "Import" function in drawing program.

Free Drawing Viewer from AutoDesk (a free, downloadable application that lets you view and print basic design drawing formats; Windows OS only)

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Model Installation Diagram

Model installation is normally accomplished through a 3x6.5 foot door in the north wall of the diffuser. Model/sting assemblies can quickly be brought into the test section via a removable ceiling track. Under special circumstances the model may be installed through the 6x9 foot ceiling panel. The model installation is presented in the image below.


Model Support System

A traversing strut downstream of the test section can be programmed to translate horizontally to maintain a desired point of rotation throughout the horizontal-plane angle-range, generally angle-of-attack.

The center of rotation in the vertical plane is 5.3 inches aft of the strut leading edge. The horizontal and vertical plane angles are continuously variable and are determined by the relative positions of a knuckle and sleeve inside the support body. The model support system can position the model at attitudes circumscribed by a 15-degree half-angle cone.

Bent primary adapters of 5, 10, 12.5, and 20 degrees are available to alter the range of model angles.

Numerous stings of differing lengths and taper arrangements are also in inventory.

Forces and Moments

Forces and moments about the model support center of rotation in the tunnel axis are limited to:


Flow Visualization

Flow visualization techniques such as Schlieren, Pressure Sensitive Paint, Oil Flow, Tufts, Sublimation, Skin Friction Interferometry, and Liquid Crystal can be obtained by appropriately positioning 2.35-foot diameter optical-quality windows in the test section sidewalls. Still and color video capability are provided.

High-Pressure Air

High-pressure air (3,000 psi) is available at weight flows up to a total of 80 pounds per second through dual, independently controlled digital valves. Air from one of these lines can be preheated using a one megawatt moveable heater.

Reflected Shock Waves

Shock waves reflecting on the model from the solid test section walls will have a detrimental effect on the model forces and pressures and must be avoided. When determining appropriate model size and attitude, calculate the shock rhombus by assuming reflections at the Mach angle from a 4-inch thick wall boundary layer.

Starting Loads

The design of models to be tested in the 9-by 7-foot Supersonic Wind Tunnel must allow for additional critical conditions associated with blockage (the ratio of model-projected frontal area to test section cross-sectional area) and transient starting loads. Large model blockages provide a potential to "unstart" the airflow, allowing a strong shock wave to pass through the test section resulting in possible damage to the model, sting and balance. The ratio of model wing span to tunnel width for minimum supersonic performance verification interference should not significantly exceed 0.5. The maximum recommended ratio of model cross-sectional area to test section cross-sectional area is about 0.010 (model at 0° angle of attack).

Normal procedure is to reduce the tunnel pressure and position the model for minimum loads before beginning the acceleration to, or deceleration from, supersonic conditions.

However, significant transient loads are still generated by the swirling, subsonic, separated flows preceding the establishment of sonic velocity in the upstream throat. To ensure that a model, sting and balance will withstand these transients, they must be designed to withstand the empirically derived starting loads indicated in the following charts.


image of Southern Ohio Real Estate Auction - 231.3± Acres in 3 ...

Southern Ohio Real Estate Auction - 231.3± Acres in 3 ...

Tract 2: 50.8± acres – income-producing tillable land, home, grain storage, and outbuildings ideal for livestock projects. The 1,320 sq. ft. ranch home with 2 bedrooms, 2 full baths, and a full basement features several modern updates and ample storage space..
From: www.thewendtgroup.com

Southern Ohio Real Estate Auction - 231.3± Acres in 3 Tracts Thursday, March 18th at 6pm

AUCTION LOCATION: New Vienna United Methodist Church, 11576 St. Rte. 28, New Vienna, Ohio. The church is located on St. Rte. 28 just ½ mile west of the intersection of St. Rte. 73 and St. Rte. 28 in New Vienna.
PROPERTY LOCATION: 5955 St. Rte. 28, New Vienna, Ohio. The farm is located on the north side of St. Rte. 28 just 1 mile east of the New Vienna Elementary School (on the Clinton-Highland county line).

Auctioneer’s Note: Wayne and Sandy are retiring from their farming operation. This provides the opportunity to bid on this large tract of land in the manner which best suits your needs.
231.3±Acres with 205± tillable acres as per FSA. The predominate soil types include Miamian silt loam, Miamian-Russell silt loam, and Xenia silt loam. Clinton County, Green Township • Highland County, Penn & Fairfield Townships • East Clinton Local School District

Inspection Dates:
Wednesday, February 17 · 4-6PM
Tuesday, March 2 · 4-6PM
A representative from The Wendt Group will be available at the home on Tract 2. Walk-overs are welcome.

Tract 1: 78.2± acres – mostly tillable income-producing cropland. Includes 1.5± acres of CRP.

Tract 2: 50.8± acres – income-producing tillable land, home, grain storage, and outbuildings ideal for livestock projects.


The 1,320 sq. ft. ranch home with 2 bedrooms, 2 full baths, and a full basement features several modern updates and ample storage space. This clean, comfortable home offers a great floorplan. Amenities include hardwood floors, bath with quarter-moon shower/tub, updated kitchen cabinets and countertops, newer windows and metal roof, 4” well, central air, and natural gas furnace. The attached 22’x23’ garage has overhead doors with an opener.


Room Dimensions:
Kitchen: 10’x14’
Dining Room: 9’x11’
Living Room: 14’x25’
Bedroom 1: 12’x15’
Bedroom 2: 11’x13’
Laundry Room: 6’x11’
Mud Room: 7’x14’
Attached Garage: 22’x23’

This tract also includes 3 outbuildings and grain storage.


· 30’x92’ 3-sided building
· 27’x76’ 3-sided building with partial concrete floor and 12’x20’ lean to
· 14’x62’ building with concrete floor and 22’x60’
    finishing floor
· (3) grain bins with drying floors and fans. Approximately
    12,000-bushel capacity.

This tract would be ideal either as an owner-occupied primary residence or as an income-producing rental property.



Tract 3: 102.3± acres – tillable and recreational land with 1.5± acres of CRP. Potential building site. Wooded land provides ideal cover for wildlife.


Annual Real Estate Taxes for the entire property: $8,700

Owners: Wayne & Sandra AmesFor more information, call :Kasey Smith 740-505-8845 or Dale Evans 260-894-0458


image of Unitary Plan Wind Tunnel 11-by 11-foot Transonic Test ...

Unitary Plan Wind Tunnel 11-by 11-foot Transonic Test ...

Feb 13, 2014 · The 11-foot TWT is a closed-return, variable-density tunnel with a fixed-geometry, ventilated test section with a flexible wall nozzle. It is one of three separate test sections powered by a common drive system. A three-stage, axial-flow compressor powered by four wound-rotor, variable-speed induction motors, produces airflow.Closed-return, variable-density tunnel with a fixed-geometry, ventilated test section with a flexible wall nozzle with a Mach range of 0.20 to 1.45..
From: www.nasa.gov

The 11-by 11-foot Transonic Wind Tunnel (11-foot TWT) Facility is part of the Unitary Plan Wind Tunnel (UPWT) complex at NASA's Ames Research Center at Moffett Field, California, where generations of commercial and military aircraft and NASA space vehicles, including the space shuttle, have been designed and tested.

The 11-foot TWT is a closed-return, variable-density tunnel with a fixed-geometry, ventilated test section with a flexible wall nozzle. It is one of three separate test sections powered by a common drive system. A three-stage, axial-flow compressor powered by four wound-rotor, variable-speed induction motors, produces airflow. Interchangeability of models among the UPWT test sections allows testing across a wide range of conditions.

The 11-foot TWT has been instrumental in the development of virtually every domestically produced commercial transport and military fixed-wing airframe since the 1960s. The facility is used extensively for airframe testing and aerodynamic studies and has played a vital role in every manned space flight program.

A model of f-18 in 11-by 11-foot Transonic Wind Tunnel at NASA's Ames Research Center.
A model of f-18 is tested in the NASA Ames 11-by 11-foot transonic wind tunnel. Image credit: NASA Ames Research Center

Adobe pdf logo  ATP 11-by 11-ft Brochure.pdf

Capability

Operating Characteristics

Test Section Dimensions

Test Section Dimensions Plan View AutoCAD Files:
 

AutoCAD logo  Version 2000i AutoCAD file (.dwg)

Translation files:

Drawing Exchange File logo  Version 2000 Drawing Exchange file (.dxf)

Initial Graphics Exchange Specification File logo  Initial Graphics Exchange Specification file(.igs)

 

*Note: Right click (PC) / control click (Mac) and "Save As..." to download files. To open, use "Import" function in drawing program.

Free Drawing Viewer from AutoDesk (a free, downloadable application that lets you view and print basic design drawing formats; Windows OS only)

Free Drawing Viewer from eDrawings (a free, downloadable application that lets you view and print basic design drawing formats; Windows and Mac)
 

Test Section Dimensions Elevation View AutoCAD Files:
 

AutoCAD logo  Version 2000i AutoCAD file (.dwg)

Translation files:

Drawing Exchange File logo  Version 2000 Drawing Exchange file (.dxf)

Initial Graphics Exchange Specification File logo  Initial Graphics Exchange Specification file (.igs)

 

*Note: Right click (PC) / control click (Mac) and "Save As..." to download files. To open, use "Import" function in drawing program.

Free Drawing Viewer from AutoDesk (a free, downloadable application that lets you view and print basic design drawing formats; Windows OS only)

Free Drawing Viewer from eDrawings (a free, downloadable application that lets you view and print basic design drawing formats; Windows and Mac)

 

Model Installation Diagram

This diagram shows a sting installed with a 40-inch extension presented in the image below.

Forces and Moments

A traversing strut downstream of the test section can be programmed to translate vertically to maintain a desired point of model-pitch rotation throughout the vertical plane angle-range. The model support center-of-rotation in the horizontal plane is 4.8 inches aft of the strut leading edge. These angles are continuously variable and are determined by the relative positions of a knuckle and sleeve inside the support body.

The model support system can position the model at attitudes circumscribed by a 15-degree half-angle cone. Bent primary adapters of 5, 10, 12.5, and 20 degrees are available to alter the range of model angles. Forces and moments resolved at the model support center of rotation are limited to:

Link to NASA Ames Balance Inventory website

Turntable Model Support

A subfloor-mounted turntable model support, used primarily for semispan model testing, is located at tunnel station 106. This support system can be rotated ± 180 degrees and forces and moments are limited to:

Semispan Testing

Provisions are available for sealing the slots in the test section floor to provide a solid image plane for semispan testing.

Installation and Personnel Access

Models can be installed through a hatch in the top of the test section. Personnel gain access to the test section through doors in the diffuser sidewalls downstream of the model support strut.

Flow Visualization

Flow visualization techniques are available through multiple, optical-quality windows in the tunnel sidewalls. Optical-quality windows are also available in the test section ceiling and floor.

High-Pressure Air

High-pressure air (3,000 psi) is available at weight flows to 80 pounds per second through dual, independently regulated lines ending within the support strut. A one-megawatt, moveable heater can preheat air from one of these lines. Preheated air at 60 pounds per second is available at the turntable.


image of OPTN is a host intrinsic restriction factor against ...

OPTN is a host intrinsic restriction factor against ...

Sep 13, 2021 · Here, Ames et al. show that Optineurin (OPTN), a conserved autophagy receptor, restricts HSV-1 spread, degrades viral VP16 through autophagy and is neuroprotective against HSV infection in vivo.Fast-replicating neurotropic herpesviruses exemplified by herpes simplex virus-1 (HSV-1) naturally infect the central nervous system (CNS). However, most individuals intrinsically suppress the virus during a primary infection and preclude it from significantly damaging the CNS. Optineurin (OPTN) is a conserved autophagy receptor with little understanding of its role in neurotropic viral infections. We show that OPTN selectively targets HSV-1 tegument protein, VP16, and the fusion glycoprotein, gB, to degradation by autophagy. OPTN-deficient mice challenged with HSV-1 show significant cognitive decline and susceptibility to lethal CNS infection. OPTN deficiency unveils severe consequences for recruitment of adaptive immunity and suppression of neuronal necroptosis. Ocular HSV-1 infection is lethal without OPTN and is rescued using a necroptosis inhibitor. These results place OPTN at the crux of neuronal survival from potentially lethal CNS viral infections. During herpesvirus infection, most individuals intrinsically suppress a primary infection and therewith preclude potential damage or neurodegeneration of the CNS. Here, Ames et al. show that Optineurin (OPTN), a conserved autophagy receptor, restricts HSV-1 spread, degrades viral VP16 through autophagy and is neuroprotective against HSV infection in vivo..
From: www.nature.com

Antibodies, stains, cells, viruses, chemicals, and plasmids

The following antibodies and stains were used in this study for imaging:

DAPI (D9542, Sigma) (1:1000), NucBlue™ Live ReadyProbes™ Reagent (Thermo Fisher, R37605) (2 drops per ml), Mouse monoclonal to LAMP1 (Abcam, ab25630, [H4A3]) (1:100), Mouse monoclonal anti-HSV1 + HSV2 VP16 (Abcam, ab110226, [LP1]) (1:100), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (Thermo Fisher, A-11030) (1:100), Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Thermo Fisher, A-21245) (1:100), Rabbit polyclonal anti- Optineurin (CTerm) (Cayman Chemical, No. 100000) (1:100), and anti-mouse CD8a APC-conjugated monoclonal antibody (Tonbo biosciences, 20-1886-U100, [clone 2.43]) (1:100).

The following antibodies were used for immunoblot:

Mouse monoclonal anti-GAPDH (Santa Cruz, sc-69778, [7B]) (1:1000), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody HRP (Thermo Fisher, 31432) (1:5000), Goat anti-Rabbit IgG (H + L) Cross Adsorbed Secondary Antibody HRP (Thermo Fisher, G-21234) (1:5000), Mouse monoclonal anti-FLAG (Sigma, F1804, [M2]) (1:1000), Mouse monoclonal anti-HSV1 ICP0 (Abcam, ab6513, [5H7]) (1:1000), Rabbit monoclonal anti-p-S177 OPTN (Cell Signaling Technologies, 57548 S) (1:1000), Mouse monoclonal anti-HSV1 + HSV2 VP16 (Abcam, ab110226, [LP1]) (1:1000), and Rabbit polyclonal anti- Optineurin (CTerm) (Cayman Chemical, No. 100000) (1:1000).

The following antibodies were used for immunoprecipitation:

Mouse monoclonal anti-FLAG (Sigma, F1804, [M2]) (5 µg), normal mouse IgG (Santa Cruz, sc-2025) (5 µg), and additionally, and Protein A/G PLUSAgarose beads (Santa Cruz, sc-2003) (20 µl) were used.

For flow cytometry the following antibodies were used:

anti-mouse CD8a APC-conjugated monoclonal antibody (Tonbo biosciences, 20-1886-U100, [clone 2.43]) (1:100). anti-mouse CD4 PE-conjugated monoclonal antibody (Tonbo biosciences, 50-0042-U100, [RM 4-5]) (1:100).

HeLa Optn−/− and parental strain were provided by Dr. Richard Youle (National Institutes of Health). Vero cells (ATCC) were used in plaque assays and in virus preparation. LUHMES cells (ATCC) were provided by Dr. David Bloom (University of Florida). HCE cells were provided by Dr. Kozaburo Hayashi (National Eye Institute, Bethesda, MD). Paul Kinchington (University of Pittsburgh) provided the dual tagged KOS strain of HSV-1 (RFP driven by gC promoter and GFP driven by ICP0 promoter).

Dr. Patricia Spear (Northwestern University) provided the KOS and 17 strains. The strain 17 γ134.5 -null mutant of HSV-1 was provided by Dr. David Leib (Dartmouth College). Dr. Prashant Desai (Johns Hopkins University) provided the HSV-1 K26-GFP KOS strain. Dr. Steven Triezenberg (Van Andel Research Institute) provided the DG1 (VP16-GFP expressing) strain of HSV-1. Gregory Smith (Northwestern University) provided the PRV-GS2484 strain (PRV-Becker expressing mRFP1-UL35 fusion protein and CMV-GFP cassette in Us4).

The following chemicals were used in this study: Bafilomycin A1 (Sigma) to inhibit autophagic flux, MG132 (Sigma) to inhibit proteasomal degradation. Cycloheximide (Sigma) to block de novo protein synthesis. Lipofectamine 2000 (Thermo Fisher) and RNAiMAX (Thermo Fisher) for transfections. Necrostatin-1s (Selleckchem) for inhibition of necroptosis. BX795 (Selleckchem) for inhibition of TBK1.

The FLAG-OPTN plasmid was provided by Dr. Beatrice Yue (University of Illinois at Chicago).

Mice

Mice used in this study were generated in the study35. In summary Optnflox/flox mice on a C57BL/6 background were generated using a targeting vector-inserted LoxP site that flanks the first coding exon and a neomycin selection cassette. Optnflox/flox mice were crossed with CMV-Cre mice (Jackson Laboratories, Bar Harbor, ME, USA) to generate CMV-Cre; Optnflox/wt mice, which were used as breeding pairs. Global Optn knockout (CMV-Cre; Optnflox/flox) mice are hereafter referred to as Optn − /− mice. 8-16 weeks old Optn − /− or Optn+/+ male and female mice were used in this study.

All experiments were performed and housed in a BSL2 rodent facility in the Biologic Resources Laboratory at the University of Illinois at Chicago with a standard 12 h light, 12 h dark cycle. Ambient temperatures at this facility are maintained between 20 and 26 °C and relative humidity between 45% and 65%. This modern animal facility has several veterinarians on staff available for expert veterinary care and advice during the project. Animal services core facility at the Department of Ophthalmology and visual sciences houses a BSL-2 facility dedicated specifically to our laboratory use. All protocols have been reviewed and certified by the animal care committee of the University of Illinois at Chicago.

Mouse infection experiments

The corneal scarification method was used for infection of mice. Mice were anesthetized using a mixture of ketamine and xylazine, and the topical anesthetic, proparacaine, was applied to the ocular surface prior to epithelial debridement. Three parallel scratches were made in the corneal epithelium with a 30-gauge needle, then mice were infected with 5 × 105 PFU HSV-1 by application of a virus containing solution to the corneal surface. For the novel object recognition test mice were infected with 1 × 105 PFU HSV-1 to reduced number of mice that reached endpoint criteria before 30 dpi. McKrae strain was used for mouse experiments. For survivorship endpoint criteria, mice had to lose >15% initial body weight rapidly, demonstrate excessive morbidity, difficulty ambulating, paralysis, or deep lesions on the head > 1cm2.

Novel Object Recognition Test

The Novel Object Recognition Test was be used to assess whether deterioration of memory is occurring in mice during ocular HSV-1 infection. Mice were 8-16 weeks old. The apparatus for testing was a white polypropylene box with a video recording camera (GoPro Hero7, GoPro) mounted above it. The base of the box is 15 inches by 20 inches and the walls of the box are 11.75″ inches high. The test for each treatment group included a familiarization session and a testing session, both of which were recorded.

The familiarization session began with cleaning the box and objects with MB10 to both disinfect the area and minimize odor cues. Each animal was subjected to a familiarization session where two identical objects were placed in the box prior to introducing the mouse to the box. The mouse was introduced to the box and allowed to explore the box for a total of 5 min. At this point the mouse was removed from the testing area and the area and objects were cleaned again with MB10 before testing the next mouse.

24 h later each animal was subjected to a testing session where two objects were placed in the box prior to introducing the mouse to the box. One object was from the familiarization session, and the second was a novel object. The session began with cleaning the box and objects with MB10 to both disinfect the area and minimize odor cues. The mouse was introduced to the box and allowed to explore the box for a total of 5 min. At this point the mouse was removed from the testing area and the area was cleaned again with MB10 before testing the next mouse.

Using the recorded footage, the time the mouse explored each object was measured manually using a stopwatch. Only exploration including closely sniffing or touching the object with whiskers or nose was counted as exploration.

Necrostatin-1s experiments

For mouse experiments, Nec-1s was administered at a low dose of 0.5 mg/kg (lo), high dose of 2.5 mg/kg (hi), or control treatment of vehicle alone (DMSO). Administration by intraperitoneal injection was performed 12 h before infection (0dpi), then daily until 7 dpi. For in vitro experiments 15 days in vitro (DIV) primary neuron culture from Optn+/+ or Optn−/− mouse embryos were infected with MOI 2.5 17 strain HSV-1 for 1 h before medium was changed. The replacement medium included 1 µg/ml propidium iodide (Invitrogen) and either 100 µg/mL Nec-1s or DMSO. Medium was used for the remainder of the experiment and images were captured using a Lionheart LX automated microscope.

BX795 experiments

106 HeLa Optn+/+ or −/− cells were plated in six-well format. The next day one group of wells were treated with 50 µM BX795 for 1 h then all groups were infected at indicated MOI with 17 strain HSV-1 for 3 h. Samples were harvested for immunoblotting.

Cycloheximide chase experiments

106 HeLa Optn+/+ or −/− cells were plated in six-well format, or 5 × 105 HCE cells were plated in 12-well format and transfected with siRNA. The next day cells were infected at MOI 1 with HSV-1 (17-strain) for 12 h. Medium (DMEM 10% FBS for HeLa, MEM 10% FBS for HCE) was replaced with fresh medium containing 100 µg/mL cycloheximide to block de novo protein translation. Samples were collected at 12 h, 18 h, and 24 h after infection for lysis in RIPA buffer and subsequent immunoblotting. In a separate experiment using only wildtype HeLa, LUHMES, or HCE cells, at 8 hpi fresh medium containing 100 µg/ml cycloheximide to block de novo protein translation and either DMSO, as a control, 200 nM Bafilomycin A1 to inhibit autophagy, or 50 µM MG132 to inhibit the proteasome. An untreated sample was taken at 8 hpi, and treated samples were taken at 24 hpi for immunoblotting.

siRNA transfection

A Dicer-Substrate Short Interfering RNAs (DsiRNAs) TriFECTa® Kit (IDT) with predesigned siRNA molecules was used for transfections in this study. Cells were plated and grown to 50% confluency. Cells were then transfected as per manufacturer’s protocol using RNAiMAX at 1 µl/mL in OptiMEM (ThermoFisher). Multiple concentrations for each premade siRNA molecule were tested and it was determined that siRNA 1 at 1 nM produced effective knockdown with minimal cell death after 48 h of transfection.

LUHMES cell culture

LUHMES cell culturing was adapted from the methods outlined36. First tissue culture flasks and plates were coated with poly-l-ornithine hydrobromide (Sigma) overnight in a biosafety cabinet then with fibronectin (Sigma) overnight in a tissue culture incubator at 37˚C. Plates were allowed to air dry before use. Proliferation medium (DMEM:F12 (ATCC) containing 1% N2 Supplement (ThermoFisher), 1X Penicillin-Streptomycin-Glutamine solution (ThermoFisher), and recombinant human FGF-basic (Fibroblast Growth Factor, Peprotech) was prepared immediately before use and was used to passage the undifferentiated LUHMES cells. Cells were plated in plates and grown to 50% confluency before differentiating in differentiation medium with 1 µg/mL tetracycline hydrochloride (Sigma), 1 mM N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (Sigma), and 2 ng/mL Glial cell-derived neurotrophic factor (GDNF). In experiments where siRNA transfection was required, the transfection was performed overnight in OptiMEM (see siRNA transfection section) prior to addition of differentiation medium. Cells were then used for experiments 4 days after differentiation.

Primary neuron culture

Embryonic brains from E18 mouse embryos were pooled for each mouse strain and digested in 5 mL 0.25% Trypsin with 75 µL 0.1% DNase (New England Biolabs) in a 37 °C water bath for 30 min. Tissue was spun down at 500 × g for 1 min to aspirate trypsin and wash pellet with 5 mL cFBS (Gibco). Cells were spun down and resuspended in 5 mL medium (485 ml Neurobasal®-A medium (Gibco), 10 mL B27 supplement (Invitrogen), 5 mL 200 mM L-glutamine (Invitrogen), 25 mM glucose). Tissue was triturated with a 5 mL plastic pipette for 10 repetitions, then with a 1000 µL pipette for 10 repetitions before being filtered through a 70 μ strainer into a new 50 mL conical tube and spun down. The medium was aspirated and the cells were resuspended in 20 mL medium. In all, 0.5 × 106 cells were plated in 1 mL medium into Poly-d-Lysine coated 24-well plates. At 3 DIV a half medium change with Cytosine β-d-arabinofuranoside hydrochloride (Ara-C) containing medium to a final concentration of 1 µM Ara-C. Subsequent restorative half medium without Ara-C were performed at DIV6, DIV8, DIV10, and DIV13. Experiments were performed on DIV15.

Viral genome isolation

Infected cell pellets were suspended in buffer containing 1% SDS, 50 mM Tris (pH 7.5), and 10 mM EDTA, and proteinase K (2 units/mL) then incubated overnight at 45 °C. Proteinase K is then heat inactivated for 30 min at 95 °C. DNA was isolated using phenol/chloroform extraction followed by ethanol precipitation. HSV-1 genomes were quantified using quantitative PCR (ABI 7500, Applied Biosystems) using HSV-1 specific primers and Fast SYBR Green Master Mix (Life Technologies). Primers are listed in Supplementary Table 1.

Time-lapse fluorescent microscopy

Immediately after the addition of HSV-1 in cell growth medium (DMEM, 10% FBS, 1% Penn/Strep), cells were placed in the environmentally controlled chamber on the stage of a Zeiss Observer spinning disk confocal microscope set to 37 °C and 5% CO2. Time-lapse experiments images were taken at 30 min intervals or 60 min intervals. Imaging was performed using either 10x or 100x objective lenses. Image analyses were performed in ZEN and imageJ software.

Fluorescent microscopy

Cells grown on 1.5 mm thick glass bottom dishes were fixed in 4% paraformaldehyde for 10 min. If antibody staining was performed, fixation was followed by permeabilization in a 0.1% Triton X 100 solution. Samples were blocked in 5% fetal bovine serum in phosphate-buffered saline (blocking buffer) for 1 h. Samples were stained with primary antibody (1:100) in blocking buffer for 1 h followed by secondary antibody (1:100) and DAPI staining for 1 h. Images were taken on a Zeiss LSM710 confocal microscope using 63x or 100x objective lenses. For the mouse brain sections, additional images were captured on a BioTek Lionheart microscope at ×10 and image-stitched using the Biotek GEN5 v3.04 Imager software.

TIRF/super-resolution microscopy

Following the same staining protocol for fluorescent microscopy with two modifications; cells were washed five times with blocking buffer after each staining, and antibody concentrations were doubled (1:50) with DAPI omitted. Imaging was performed using a DMi8 S platform equipped for TIRF and ground state depletion (GSD) (Leica). OxEA buffer, an oxygen scavenger buffer, must be made immediately before use and is exhausted after ~1 h of imaging. OxEA (50 mM β-MercaptoEthylamine hydrochloride [Sigma], 3% v/v OxyFluor [Oxyrase Inc], 20% v/v sodium dl-lactate solution [Sigma], in PBS pH 8-8.5 adjust with NaOH) was added to the fixed, permeabilized, and stained cells prior to imaging. Once a location of interest was TIRF-imaged using the LAS X software to calculate and adjust the machine for a 250 nm depth image, GSD was used starting with the longest excitation wavelength and ending with the shortest to collect 5000 images of “blinking” fluorescence for each channel. Post-imaging dSTORM reconstruction in FIJI with the ThunderSTORM plugin was performed to acquire super-resolution images.

Immunohistochemistry

Tissue was embedded in OCT Compound (Thermo Fisher Scientific) and frozen. 10 µm sections were cut from the tissue in a CryoStar NX50 (Thermo Fisher Scientific) cryotome and collected on double frosted slides (Thermo Fisher Scientific). Slides were fixed in cold 100% acetone for 10 min. Slides are then washed three times in phosphate-buffered saline with tween-20 (PBST). Slides were blocked in 5% Bovine Serum Albumin in PBS for 30 min at room temperature. Slides were then stained with the primary antibody (1:100 in PBST) for 60 min at room temperature followed by three washes in PBST. Protected from light, slides were stained with the secondary antibody (1:100 in PBST) and DAPI for 60 min at room temperature. Slides were washed three more times in PBST then mounted Vectashield hardset mounting medium (Vector Laboratories, Burlingame, CA, United States), sealed with clear nail polish, then visualized by fluorescence microscopy. For TUNEL staining, cyrosections of mouse brain tissue was stained using a TUNEL Assay Kit - HRP-DAB (ab206386) (Abcam) according to manufacturer’s protocol. For HSV-1 VP16 and gB stainings, the M.O.M.® (Mouse on Mouse) Immunodetection Kit, Fluorescein (FMK-2201) (Vector Laboratories, Burlingame, CA, United States) was used according to manufacturer’s protocol.

Immunohistochemical detection of optineurin in human nervous systemtissues

Human nervous system tissues analyzed by immunohistochemistry for optineurin expression were derived from autopsies performed at the University of Illinois at Chicago. Autopsies were obtained after informed consent and tissue use conformed to Institutional Review Board (IRB)-approved protocols. Sections of paraffin-embedded human brain tissues were prepared and evaluated using standard histopathologic techniques and neuropathologic diagnostic criteria by a board-certified neuropathologist. For immunohistochemical analysis, 5 µm sections of paraffin-embedded formalin fixed tissue were deparaffinized with xylene and rehydrated. Antigen unmasking was performed in 1X citrate buffer, pH 6.0 just below boiling for 10 min. Reaction container and slides were allowed to cool for 20 min. The slides were then removed from reaction container, washed in dH2O, incubated in 3% hydrogen peroxide for 10 min, washed in dH2O, and washed in PBS for 5 min. Immunohistochemisty was performed using the Vectastain Elite ABC-HRP Kit (Rabbit IgG) (PK-6101) using a 1:300 dilution of Optineurin C-TERM rabbit polyclonal antibody (Cayman). Detection was achieved by incubating slides in ImmPACT DAB (SK- 4105). Counterstaining was performed by incubating slides in a 50% hematoxylin solution for 30 s. Slides were then dehydrated with ethanol and mounted with Permount.

Flow cytometry

Cells were removed from culture plate using HANK’s dissociation buffer. Cells were washed in PBS with 5% FBS then fixed with 4% paraformaldehyde. Cell were washed again in PBS 5% FBS then resuspended in the same buffer for flow cytometry on an Accuri C6 flow cytometer (BD Sciences). Data collection was performed using the BD Accuri C6 Plus v1.0 software. Histograms were prepared in FlowJo v10.0.7 software. For brainstem analysis, tissue was dissociated in 100 µL of 5 mg/mL collagenase IV (Sigma Aldrich) in OptiMem (Gibco). The tissue was spun down at 500 × g for 1 min and resuspended in FACS buffer (PBS, 1% BSA). The cell suspension was filtered using 100 µm sterile filter, then blocked using TruStain FcX (BioLegend) according to manufacturer’s protocol. After 2 rounds of centrifugations and washes in 1 mL FACS buffer, the samples were stained for CD3, CD4, and CD8 using 1 µL of antibody and 100 µL (10%) of the cell suspension for 1 h. After 2 rounds of centrifugations and washes in 1 mL FACS buffer, the samples were resuspended in 1 mL FACS buffer and immediately analyzed on an Accuri C6 flow cytometer (BD Sciences).

Apoptosis detection by flow cytometry

Optn+/+ and Optn−/− HeLa cells were infected with HSV-1 at 0.1 MOI and harvested at 24 hpi. FITC Annexin V/Dead Cell Apoptosis Kit (InvitrogenTM) was used to detect apoptosis. As per the manufacturer protocol the sample pellets were washed with cold PBS and then suspended in 1x annexin binding buffer. The suspension was mixed with 5 µL FITC Annexin V and 1 µL of a propidium iodide solution (100 µg/mL) for 15 min in the cold. The mixture was diluted with 1X annexin binding buffer. Further flow cytometry (BD AccuriTM C6 Plus flow cytometer) was used to analyze the staining for Annexin V and propidium iodide. Flow data was analyzed using FlowJo software (Tree Star Inc.).

Plaque assays

After scraping cells from a culture dish cell pellets were resuspended in 1 mL of Opti-Mem. Cell suspension were sonicated for a 5 s pulse at 25% amplitude then immediately serially diluted in Opti-MEM. For tissue samples, the tissue was place in 1 ml of Opti-MEM and sonicated as described. Dilutions are added to Vero cell monolayers and incubated at 37 °C and 5% CO2 for 2 h. Following incubation the virus dilution is aspirated and fresh medium (DMEM, 10% FBS, 1% Penn/Strep, 5% methylcelluose w/v) is added to the culture dish. Cells are returned to incubator (37 °C and 5% CO2) for ~3 days or until plaques can be observed. Monolayers are fixed by adding methanol directly to the medium for 10 min at room temperature and then aspirating the methanol-containing medium. The cells are stained with crystal violet (70% water, 20% ethanol, 10% crystal violet stock [1 g/100 mL crystal violet in 20% ethanol]) for 1 h, then aspirated and dried to visually count plaques under a microscope.

Immunoprecipitation

Cells were scraped from culture dishes and centrifuged to pellet cells. Cell pellets were lysed on ice for 1 h in immunoprecipitation (IP) buffer (250 mM NaCl, 50 mM Tris, 0.5 mM EDTA, 0.5% NP-40) with protease/phosphatase inhibitor added. For the anti-VP16 immunoprecipitation, 10 mM N-Ethylmaleimide was added to the IP Buffer immediately before use. Lysates were centrifuged to remove cell debris and the soluble portion was precleared with Isotype control antibody (Santa Cruz) and protein A/G conjugated beads (Santa Cruz) for 1 h with agitation at 4 °C. Beads were pelleted by centrifugation and lysates were moved to new tubes. The lysates were then incubated with isotype or specific antibody (5 µg per 1.7 mL tube with 1 mL lysate) on ice for 1 h. 20 µL of protein A/G beads were added and samples were agitated at 4 °C overnight. The beads containing the immunoprecipitated proteins were pelleted by centrifugation and the unbound portion decanted. The beads were washed four times in IP buffer prior to processing for immunoblot analysis.

Immunoblot

Cells were scraped from cell culture dish and cells collected in 1.7 mL tubes were centrifuged. Cell pellets were lysed on ice for 30 min using RIPA buffer (Sigma) with added protease and phosphatase inhibitor unless otherwise stated for immunoprecipitation. Lysates were centrifuged to pellet debris and the soluble fraction was mixed with NuPage LDS Sample Buffer (Life Technologies) and β-mercaptoethanol. Samples were incubated on a heat block at 95 °C for 10 min. Electrophoresis was performed using a NuPage 4–12% gradient polyarcrylamide gel (Life Technologies) with NuPage MOPS running buffer (Life Technologies) at 70 V at room temperature. Proteins were transferred to a PVDF membrane using an iBlot 2 system (Thermo Fisher Scientific). Membranes were blocked for 1 h in blocking buffer (5% milk in tris-buffered saline with 0.1% tween-20 (TBST)) followed by overnight primary incubation (1:1000–1:5000 in blocking buffer) with gentle rocking at 4 °C. Membranes were washed three times in TBST before incubation with secondary anti-rabbit or anti-mouse antibody conjugated to HRP (1:10,000 in blocking buffer) for 2 h at room temperature. ECL Femto Substrate (Thermo Fisher Scientific) was used to develop blots and bands were visualized using a Quant 4000 (General Electric) and densitometry analysis was performed in ImageQuant TL and ImageJ software. GAPDH was used as a reference control for sample loading.

Entry assay

In all, 2 × 104 cells per well were plated in a 96-well format. Cells were confluent after incubation overnight and were then infected at multiple MOIs with gL86 HSV-1. 6 hpi the medium was aspirated, and the cells were washed with PBS. In all, 100 µL of β-galactosidase substrate (0.5% NP40, 3 mg/mL O-nitro-phenyl-β-d-galactopyranoside [Thermo Fisher Scientific]) was added to each well and plated were incubated at 37 °C for 2 h. Colorimetric reaction signal was measured at 410 nm using a GENESIS Pro Plate reader. Alternatively, cells were plated in a 12-well format and infected with gL86 HSV-1 at the same MOIs for the same amount of time. X-gal was used to develop the reaction and the plate was scanned to compare the color visually.

Infections

Infections were performed by suspending HSV-1 from a thawed stock in Opti-MEM and adding this mixture to cell cultures. Cells are incubated with the virus for 2 h before medium is removed and virus-free growth medium is added to cells. In total, 17 strain was used for in vitro experiments unless otherwise specified.

Transfection

HeLa cells were plated to be 60–70% confluent in six-well format. In all, 2 µL Lipofectamine 2000 (Thermo Fisher Scientific) was used to transfected 2 µg of plasmid as directed by the manufacturer’s protocol in Opti-MEM. In total, 6 h after transfection cells were washed in growth medium and then incubated for 18 h (24 h total). Cells were transferred to imaging dishes or lysates were collected for experimental use.

Image analysis

Image analysis was performed in ImageJ software. For time-lapse imaging ImageJ was used to set a color threshold for intense green fluorescence. This threshold allowed the selection and measurement of the infected area relative to total area for each frame, and this data was subsequently graphed in GraphPad Prism. The infection velocity was derived from the change in the infected area between two adjacent time-points, and graphed.

Particle counts were collected in ImageJ by setting color thresholds for each particle type, then using the particle analysis feature in ImageJ to count the number of particles present in the frame.

Colocalization was analyzed in ImageJ. Background fluorescence was subtracted from the raw data of each individual channel image using a rolling ball method with a radius of 30 pixels. Following subtraction, colocalization was quantified by finding the Mander’s correlation coefficient (MCC) using the Coloc 2 plugin. The region of interest used was hand-drawn around the cell or the nucleus to determine the MCC for the nucleus or extranuclear region.

Localization of OPTN was analyzed in ImageJ by similarly subtracting background and drawing regions of interest in the raw data images. The selected regions were measured for signal area and total area and these measurements were used to determine the signal density for the nucleus or extranuclear region.

Cytokine profiling

Brainstem or draining lymph node samples were collected from mock or 8 dpi mice and profiled using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay (MCYTOMAG-70K, Millipore Sigma), with the aid of a Millipore Sigma field application scientist. The measurements were analyzed using a K-means clustering algorithm and plotted as heatmaps using the MIT Broad Institute’s Morpheus online tool (https://software.broadinstitute.org/morpheus/).

Statistics and reproducibility

All statistical analysis and graph making was carried out in GraphPad Prism software, except for flow cytometry histograms and dot plots produced in FlowJo software. Error bars represent ± SEM of at least three independent measurements (n = 3). Asterisks denote a significant difference, as determined by two-tailed unpaired Student’s t test, Mann–Whitney U test, or Logrank test: p < 0.05; p < 0.01; p < 0.001; ns not significant, or two-way ANOVA; p < 0.0001. All experiments were repeated independently a minimum of three times.

Ethical approval

The authors have complied with all regulations regarding the use of research animals and the study protocol was approved by the University of Illinois at Chicago Animal Care Committee (protocol: 20-065).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.


image of Safety Evaluation of Soy Leghemoglobin Protein Preparation ...

Safety Evaluation of Soy Leghemoglobin Protein Preparation ...

Apr 11, 2018 · The leghemoglobin protein (LegH) from soy (Glycine max) expressed in Pichia pastoris (LegH preparation, LegH Prep) imparts a meat-like flavor profile onto plant-based food products.The safety of LegH Prep was evaluated through a series of in vitro and in vivo tests. The genotoxic potential of LegH Prep was assessed using the bacterial reverse mutation assay …The leghemoglobin protein (LegH) from soy (Glycine max) expressed in Pichia pastoris (LegH preparation, LegH Prep) imparts a meat-like flavor profile onto plant-based food products. The safety of LegH Prep was evaluated through a series of in vitro and ....
From: www.ncbi.nlm.nih.gov

The leghemoglobin protein (LegH) from soy (Glycine max) expressed in Pichia pastoris (LegH preparation, LegH Prep) imparts a meat-like flavor profile onto plant-based food products. The safety of LegH Prep was evaluated through a series of in vitro and in vivo tests. The genotoxic potential of LegH Prep was assessed using the bacterial reverse mutation assay (Ames test) and the in vitro chromosome aberration test. LegH Prep was nonmutagenic and nonclastogenic in each test, respectively. Systemic toxicity was assessed in a 28-day dietary study in male and female Sprague Dawley rats. There were no mortalities associated with the administration of LegH Prep. There were no clinical observations, body weight, ophthalmological, clinical pathology, or histopathological changes attributable to LegH Prep administration. There were no observed effects on male reproduction in this study, but the suggestion of a potential estrous cycle distribution effect in female rats prompted a second comprehensive 28-day dietary study in female Sprague Dawley rats. This study demonstrated that female reproductive parameters were comparable between rats treated with LegH Prep and concurrent control rats. These studies establish a no observed adverse effect level of 750 mg/kg/d LegH, which is over 100 times greater than the 90th percentile estimated daily intake. Collectively, the results of the studies presented raise no issues of toxicological concern with regard to LegH Prep under the conditions tested.

In this study, we evaluated the safety of LegH Prep with both in vitro and in vivo models. To evaluate potential genotoxicity of LegH Prep, a bacterial reverse mutation test (Ames test) and an in vitro chromosome aberration test were performed. These in vitro models showed that LegH Prep is neither mutagenic nor clastogenic. Systemic toxicity was evaluated by a 28-day feeding study in Sprague Dawley rats. An additional 28-day feeding study was performed to evaluate the female estrous cycle and reproductive health. These in vivo models demonstrated no adverse effects attributable to the consumption of LegH Prep at the maximum dose tested, which was more than 100 times greater than the 90th percentile estimated daily intake (EDI) in ground beef analogue products. Collectively, the results of the studies presented raise no issues of toxicological concern with regard to LegH Prep under the conditions tested.

In pursuit of the intended use in ground beef analogue products, the gene encoding a soy LegH was introduced into the genome of the yeast Pichia pastoris, enabling the production of high levels of soy LegH preparation (LegH Prep). The total protein fraction of LegH Prep contains at least 65% LegH. The remaining proteins are from the Pichia host. Pichia pastoris is nontoxigenic and nonpathogenic and has been used in the recombinant expression of both Generally Recognized as Safe (GRAS) and US Food and Drug Administration (FDA)-approved proteins.28,29 Previously, the LegH and Pichia proteins within LegH Prep were evaluated for potential risk of allergenicity and toxicity in accordance with the CODEX Alimenterius Commission 2003/2009 guidelines for genetically modified foods and for novel food ingredients.30 The analysis included literature search, sequence homology comparison to known allergens and toxins, and sensitivity to pepsin digestion in a simulated gastric fluid. There was no evidence in the scientific literature to suggest allergenicity or toxicity for these proteins, and the LegH protein sequence did not contain significant homology to any known allergens or toxins. Although some of the proteins from the Pichia host present within LegH Prep had modest identity matches to minor airway allergens and/or proteins from toxic organisms, these proteins have analogous identity matches to proteins from the widely consumed yeast Saccharomyces cerevisiae, which has no evidence of food allergy or toxicity. Leghemoglobin protein and the Pichia proteins within LegH Prep were readily digested in simulated gastric fluid. Collectively, the authors of this study concluded that there was no evidence to suggest that food products containing LegH Prep posed any significant risk of dietary allergy or toxicity to consumers.30

In animal-derived meat, upon cooking, myoglobin, a heme protein exceptionally abundant in animal muscle tissue, unfolds and exposes the heme cofactor. The cofactor then catalyzes a series of reactions that transform the amino acids, nucleotides, vitamins, and sugars naturally found in animal muscle tissue, into a highly specific and diverse set of hundreds of flavor and aroma compounds, the combination of which create the unmistakable and distinctive flavor profile of meat. In plant-based meats, the leghemoglobin protein (LegH) from soy (Glycine max), a close structural ortholog of myoglobin, performs a crucial, parallel role: It unfolds upon cooking, releasing its heme cofactor to catalyze reactions that can transform the same ubiquitous biomolecules, isolated from plant-based sources, into the array of compounds that comprise the unique flavor and aroma of meat.11 Although the primary amino acid sequence of LegH is highly divergent from the sequence of animal hemoglobins and myoglobins, the 3-dimensional structure is highly similar.26 Additionally, the heme cofactor bound to LegH (heme B) is identical to heme found in animal meat, which has a long history of safe use in the human diet.14 The iron from LegH has an equivalent bioavailability to iron from bovine hemoglobin when supplemented in a food matrix.27 However, because soy root nodules, and thus LegH, are not commonly consumed in the human diet, the safety properties of LegH remain not fully understood.

An investigation of the molecular mechanisms underlying the unique flavors and aromas of meat led to the discovery that heme is the critical catalyst of the chemical reactions that transform simple biomolecules into the complex array of odorants and flavor molecules that define the characteristic flavor profile of meat.11 Heme is an iron-containing porphyrin ring that exists as a protein cofactor in all branches of life and is essential for most biochemical processes involving molecular oxygen.12 Myoglobin and hemoglobin proteins from animal meat tissues have been consumed throughout human history and represent an important source of dietary iron.13,14 Plants contain symbiotic and nonsymbiotic hemoglobin proteins, both of which share a common ancestor with animal hemoglobins.15 Symbiotic plant hemoglobins, also known as leghemoglobins, are present in the root nodules of leguminous plants.16 Leghemoglobin controls the oxygen concentration in the area surrounding symbiotic nitrogen-fixing bacteria.15–17 Nonsymbiotic plant hemoglobins are expressed in the stems, roots, cotyledon, and leaves and are involved in oxygen homeostasis pathways.18,19 Due to the presence of nonsymbiotic hemoglobins in legumes, cereals, and other plants,12,15,20–22 low levels of plant heme proteins are widely consumed in the human diet.23–25

Western diets containing meat have a larger negative impact on the environment compared to more sustainable plant-based diets.1–4 However, due to both social and personal reasons, many consumers are reluctant to reduce the amount of meat they eat.2,5,6 To date, plant-based diets have been limited to small populations, such as consumers who follow vegetarian or vegan principles.7–9 One potential way to catalyze widespread shift to more sustainable, plant-based diets is to create meat directly from plants that satisfies the tastes of meat consumers.7,8,10 Achieving that goal would require products that recreate the sensory properties that people crave in meat, including texture, mouthfeel, taste, smell, and cooking experience, based on an understanding of the biochemical origins of meat sensory attributes.

Mean and standard deviations (SDs) were calculated for all quantitative data. For the Chromosome Aberration Study, the Fisher exact test was used to compare the induction of chromosome aberrations in treated cultures and solvent control. Significance was judged at a probability value of P < 0.05. Male and female rats were evaluated separately. Body weights, food consumption, UVOL, hematology, BLD chemistry, absolute and relative organ weights, averages, and standard deviations were calculated and analyzed by Bartlett test for homogeneity of variances and normality.47 Where Bartlett test indicated homogeneous variances, treated and control groups were compared using a 1-way analysis of variance (ANOVA). When ANOVA was significant, a comparison of the treated groups to control by Dunnett test for multiple comparisons was performed.48,49 Where variances were considered significantly different by Bartlett test, groups were compared using a nonparametric method (Kruskal-Wallis non-parametric ANOVA).50 When nonparametric ANOVA was significant, comparison of treated groups to control was performed using Dunn test.51 Statistical analysis was performed on all quantitative data for in-life and organ weight parameters using Provantis Version 8, Tables and Statistics, Instem LSS, Staffordshire United Kingdom. Clinical pathology was preliminarily tested via Levene test52 for homogeneity and via Shapiro-Wilk test53 for normalcy followed by ANOVA followed with Dunnett test.48,49

This study was conducted at PSL, and the protocol was approved by the IACUC of PSL. Adult CRL Sprague-Dawley CD IGS female rats were purchased from Charles River Laboratories and subsequently quarantined and acclimated at the PSL facilities as described above. Sixty rats were selected for testing using the acceptance criteria described above, and animals were distributed into 4 groups with 15 females per group (1 control group and 3 dietary dose levels). Freeze-dried LegH Prep was administered in the diet at concentrations that targeted 0 (group 1, control), 250 (group 2, low), 500 (group 3, medium), and 750 (group 4, high) mg/kg/d of the soy leghemoglobin active ingredient (Supplemental Table S12). Food consumption was determined as described above. The estrous cycle was evaluated daily by vaginal cytology for a period of 14 days prior to administration of the test substance and for the last 2 weeks of the 28-day period of test substance administration. Estrous cycle stage was not evaluated for the first 2 weeks of the dosing period to avoid overmanipulating the animals. For each 14-day period, average estrous cycle length was calculated for each animal and subsequently each group. Clinical observations, food consumption, body weight, and food efficiency were evaluated as described above. All animals were subjected to a full necropsy, which included examination of the external surface of the body, all orifices, and the thoracic, abdominal, and cranial cavities and their contents. Female reproductive organs were collected and preserved in 10% neutral buffered formalin. The ovaries with oviducts and uterus were weighed wet as soon as possible after dissection to avoid drying. Reproductive tissues were fixed and examined as described above. Estrous cycle stage was recorded for all animals. Histological examination was performed on the preserved organs and tissues for group 1 and group 4 animals. Slide preparation was performed by Histoserv Inc (Germantown, Maryland), and histopathological assessment was performed by a board certified veterinary pathologist at Regan Path/Tox Services.

Prior to study initiation and again on study day 23, the eyes of all rats were examined by focal illumination and indirect ophthalmoscopy. Clinical observations, food consumption, body weight, and food efficiency were evaluated as described above. On study day 22, samples were collected from all animals for hematology, serum chemistry, and urinalysis evaluation. Blood samples for hematology and serum chemistry were collected via sublingual bleeding under isoflurane anesthesia. Approximately 500 μL of BLD were collected in a precalibrated tube containing K2EDTA for hematology assessments. The whole BLD samples were stored under refrigeration and shipped on cold packs. Approximately 1000 μL of BLD were collected into a tube containing no preservative for serum chemistry assessments. Hematology included RBC, HCT, MCH, ARET, total WBC, and differential leukocyte count, MCHC, HGB, MCV, RDW, and PLT. Coagulation included prothrombin time and activated partial thromboplastin time (APTT). Clinical chemistry included serum aspartate amino transferase, sorbitol dehydrogenase, total bilirubin, BLD creatinine, triglycerides, total serum protein, globulin (GLOB), inorganic phosphorus, potassium (K), serum alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, total cholesterol, fasting glucose (GLUC), albumin (ALB), calcium (CALC), sodium (Na), and chloride (Cl). Urinalysis included, quality, color (COL), clarity, urine volume (UVOL), microscopic urine sediment examination, pH, GLUC, specific gravity, protein (UMTP), ketone, bilirubin, blood (BLD), and urobilinogen. At terminal killing, all animals were euthanized by exsanguination from the abdominal aorta under isoflurane anesthesia, and BLD was collected for evaluation of coagulation parameters. All clinical pathology samples were sent to DuPont Haskell Global Centers for Health and Environmental Sciences for analysis. All animals in the study were subjected to a full necropsy, which included examination of the external surface of the body, all orifices, and the thoracic, abdominal, and cranial cavities and their contents. Tissues/organs representing systems were collected and preserved in 10% neutral-buffered formalin with the exception of the eyes, testes, and epididymides, which were preserved in Davidson’s fixative before transfer to ethanol. A subset of tissues/organs were weighed wet as soon as possible after dissection to avoid drying including adrenal glands, kidneys, spleen, brain, liver, thymus, testes, epididymides, ovaries with oviducts, uterus, and heart. The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin, and examined by light microscopy. Histological examination was performed on the complete set of preserved organs and tissues of the animals from the vehicle control and high-dose groups (groups 1 and 4, respectively) as detailed in the OECD 408 guidelines, with the exception of the female reproductive organs, which were evaluated at all dose levels. Slide preparation and histopathological assessment was performed by a board-certified veterinary pathologist at HSRL. A pathology peer review was performed by a board-certified veterinary pathologist at Regan Path/Tox Services (Ashland, Ohio) for all female reproductive tissues.

This 28-day feeding study was conducted at PSL in accordance with GLP and follows OECD Guidelines for Testing of Chemicals, Section 4 Health Effects (Part 408): Repeated Dose 90-day Oral Toxicity Study in rodents40,46 and the US FDA Toxicological Principles for the Safety Assessment of Food Ingredients, IV.C.4.a.44 and was approved by the IACUC of PSL. Adult CRL Sprague-Dawley CD IGS rats were purchased from Charles River Laboratories and subsequently quarantined and acclimated to the PSL facilities as described above. Eighty rats were selected for testing, using acceptance criteria described above, and distributed into 4 groups with 10 males and 10 females per group (1 control group per sex and 3 dietary dose levels per sex). The freeze-dried LegH Prep was administered in the diet at concentrations that targeted 0 (group 1, control), 250 (group 2, low), 500 (group 3, medium), and 750 (group 4, high) mg/kg/d of the soy leghemoglobin active ingredient (Supplemental Table S11). Food consumption was determined as described above.

The animals were observed for viability, signs of gross toxicity, and behavioral changes at least once daily during the study and weekly for a battery of detailed observations. Body weights were recorded 2 times during the acclimation period (including prior to dosing on study day 1) and on study days 3, 7, 10, and 14. Individual food consumption, based on total food consumed per cage divided by 2 to account for paired housing, was also recorded to coincide with body weight measurements. Food efficiency was calculated by dividing the mean daily body weight gain by the mean daily food consumption. The animals were fasted overnight prior to BLD collection. Samples were collected from all animals for hematology evaluation via the inferior vena cava under isoflurane anesthesia during the necropsy procedure. Approximately 500 μL of BLD were collected in a precalibrated tube containing dipotassium ethylenediaminetetraacetic acid (K2EDTA). All clinical pathology samples were sent to DuPont Haskell Global Centers for Health and Environmental Sciences (Newark, Delaware) for analysis. Clinical pathology included the following hematology analyses: erythrocyte count (RBC), hematocrit (HCT), mean corpuscular hemoglobin (MCH), absolute reticulocyte count (ARET), total white BLD cell (WBC), and differential leukocyte count, MCH concentration (MCHC), hemoglobin concentration (HGB), mean corpuscular volume (MCV), red cell distribution width (RDW), and platelet count (PLT). Gross necropsy was performed on study day 15, and the animals were evaluated for any macroscopic changes. Histological examination was performed on the liver, spleen, and bone marrow of the animals from the vehicle control and high dose (groups 1 and 4, respectively). Slide preparation was performed by Histo-Scientific Research Laboratories (HSRL, Mount Jackson, Virginia), and histopathological assessment was performed by a board-certified veterinary pathologist at PSL.

This study was conducted at PSL following the OECD 407 Guidelines for Testing of Chemicals43 and Food Ingredients and US FDA Toxicological Principles for the Safety Assessment of Food Ingredients21 IV.C.4.a44 and was approved by the Institutional Animal Care and Use Committees (IACUC) of PSL. PSL is Association for Assessment and Accreditation of Laboratory Animal Care accredited and certified in the appropriate care of all live experimental animals and maintains current staff training ensuring animals were handled humanely during the experimental phase of this study in compliance with the National Research Council’s 2011 Guide for the Care and Use of Laboratory Animals (8th ed.).45 Charles River Laboratories (CRL) Sprague-Dawley CD International Genetic Standardization (IGS) rats were purchased from Charles River Laboratories (Kingston, New York) and subsequently quarantined and acclimated to the PSL facilities. Animals were maintained in a temperature- and humidity-controlled room at 19°C to 22°C and 41% to 65%, respectively, under a 12-hour light–dark cycle and fed a standard Envigo Teklad Global 16% Protein Rodent Diet #2016 (Envigo Laboratories, Inc, Indianapolis, Indiana). The diet and filtered tap water were supplied ad libitum. All contaminants within the diet and filtered tap water were within acceptable regulatory standards. The animals were housed in pairs and received enrichment activities such as chew sticks throughout the duration of the study. Forty-eight animals were selected for the test (7-8 weeks of age at dosing; weighing 230-264 g [males] and 158-181 g [females]) and distributed into 4 groups with 6 males and 6 females each (1 control group per sex and 3 dietary levels per sex). The freeze-dried LegH Prep was administered in the diet at concentrations that targeted 0 (group 1, control), 125 (group 2, low), 250 (group 3, medium), and 500 (group 4, high) mg/kg/d of the soy leghemoglobin-active ingredient (Supplemental Table S10). Food consumption was determined by biweekly feed jar weight as well as weighing any feed that was spilled into the cage. For food consumption measurements, consumed feed weight was divided by 2 to account for paired housing.

In addition to the mitotic index determination, the proliferation index of selected samples (negative control and high doses of LegH Prep) was calculated using the BrdU (5-bromo-2’-deoxyurindine) technique. The proliferation index was calculated using Equation 1 (where M1 is the first generation, M2 is the second generation, and M3 is the third generation) based on the number of cell divisions undertaken during the experiment.

The chromosomal aberration assay was conducted at Eurofins Biopharma (Munich, Germany) in compliance with the German GLP regulations according to 19b Abs. 1 chemikaliengesetz39 and the protocol procedures described in the Term Tests for Genetic Toxicity and OECD 473, In Vitro Mammalian Chromosome Aberration Test40,41 and the European Commission Regulation (EC) no440/2008 B.10.42 The study was conducted using human peripheral blood (BLD) lymphocytes (HPBL) in both the absence and the presence of the chemically induced rat liver S9 metabolic activation system (Trinova Biochem, Giessen, Germany). Peripheral BLD lymphocytes were obtained from healthy nonsmoking donors who had no recent history of exposure to genotoxic chemicals and radiation. Peripheral BLD lymphocytes were cultured in complete medium (Roswell Park Memorial Institute (RPMI) 1640 containing 15% heat inactivated fetal bovine serum, 0.24 g/mL of phytohemagglutinin, 100 units penicillin, and 100 µg/mL streptomycin). The cultures were incubated under standard conditions (37°C in a humidified atmosphere of 5% CO2 in air) for 48 hours. The cells were treated for periods of 4 or 24 hours in the nonactivated test system and for a period of 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation. Cyclophosphamide and ethylmethanesulfonate (Sigma-Aldrich, Missouri) were evaluated as the concurrent positive controls for treatments with and without S9, respectively.

The confirmatory test employed the preincubation modification of the plate incorporation test. The test or control substances, bacteria suspension, and S9 (or substitution buffer) were incubated under agitation for approximately 30 minutes at approximately 37°C ± 2°C prior to mixing with the overlay agar and pouring onto the minimal agar plates before proceeding as described for the initial test. Following incubation, the revertant colonies were counted manually and/or with the aid of a plate counter (Colony Plate Reader: Model Colony-Doc-It, Colony Doc-itTM 125 Imaging station UVP, P/N 97-0539-01). To be considered valid, the background lawn for vehicle control plates had to appear slightly hazy with abundant microscopic nonrevertant bacterial colonies. The mean revertant colony counts for each strain treated with the vehicle had to lie close to or within the expected range, taking into account the laboratory historical control range. For each experimental point, the mutation factor (MF) was calculated by dividing the mean revertant colony count by the mean revertant colony count for the corresponding concurrent vehicle control group. The results were considered to be positive when the MF was increased at least by a factor of 2 for strains TA98, TA100, and WP2 uvrA or by at least a factor of 3 for strains TA1535 and TA1537. In addition, any increases had to be dose related and/or reproducible, that is, increases must be obtained at more than 1 experimental point (at least 1 strain, more than 1 dose level, and more than 1 occasion or with different methodologies).

Plates were prepared in triplicate and uniquely identified. Appropriate sterility control check plates (treated with critical components in the absence of bacteria) were included as a standard procedural check. After pouring, the plates were placed on a level surface until the agar gelled, then inverted, and incubated at 37°C ± 2°C until growth was adequate for enumeration (approximately 65 ± 3 hours).

The Ames test (reverse mutation test) was performed by Product Safety Labs (Product Safety Labs (PSL); Dayton, New Jersey) and was conducted in accordance with US FDA good laboratory practice (GLP) regulations (21 CFR Part 58). The assay design was based on The Organization for Economic Co-operation and Development (OECD) guideline 47137 and ICH Guidelines S2A and S2B.38 Five bacterial strains were evaluated (Salmonella typhimurium (ST) TA98, TA100, TA1535, and TA1537 and Escherichia coli (EC) WP2uvrA; Molecular Toxicology, Inc, Boone, North Carolina) according to the plate incorporation and preincubation methods both in the presence and in the absence of a metabolic activation system (S9 mix). Sterile water served as the negative control and as the vehicle, while 5 mutagens including sodium azide (NaN3), ICR 191 acridine, daunomycin, methyl methanesulfonate, and 2-aminoanthracene (2-AA; Molecular Toxicology, Inc, Boone, North Carolina) were used as the positive controls. Water was also used as the solvent for the positive controls except for 2-AA, which was prepared in dimethyl sulfoxide. The initial test followed the plate incorporation method in which the following materials were mixed and poured over the surface of a minimal agar plate: 100 μL of the prepared test solutions, negative (vehicle) control, or prepared positive control substance; 500 μL S9 mix or substitution buffer; 100 μL bacteria suspension (ST or EC); and 2000 μL overlay agar maintained at approximately 45°C.

Ground beef analogue products will be formulated to contain approximately the same amount of heme as beef. This equates to a typical and maximum usage rate of 0.6% and 0.8% LegH, respectively. The EDI for LegH within ground beef analogue products was calculated based on 100% capture of the US ground beef market, which is approximately 500 times higher than the market size for all meat and poultry analogue products (note 1). The national mean daily consumption of ground beef for males and females aged 2 and older is 25 g/day (59 g beef/person/d × 42% of beef sales are ground beef).35,36 Replacement of ground beef with ground beef analogue on a one-for-one basis would result in typical (0.6% LegH use rate) and maximum (0.8% LegH use rate) Leghemoglobin protein EDIs of 150 and 200 mg/person/d, respectively. In accordance with FDA guidelines, the 90th percentile EDI was calculated as 2 times the maximum EDI or 400 mg/person/d LegH, which corresponds to 6.67 mg/kg bodyweight/day assuming an average body weight of 60 kg. The 90th percentile was used as a basis for safety testing.

During each of the in vivo studies described below, the LegH concentrations within the neat test substance and animal feed samples were analyzed by Impossible Foods using high-performance liquid chromatography (HPLC) to evaluate test substance concentration, stability, and homogeneity. Leghemoglobin protein preparation was extracted from the feed by adding 50 mmol/L potassium phosphate pH 7.4, 150 mmol/L sodium chloride to each feed sample followed by 1 hour of end-over-end rotation. High-performance liquid chromatography was performed using an Agilent 1100 Series instrument with an ACQUITY xBridge BEH125 SEC 7.8 × 150 mm ID 3.5 μm column (Waters, Milford, MA). Leghemoglobin protein concentration was determined by integration of the 415 nm absorbance at the LegH retention time.

Leghemoglobin protein was recombinantly expressed in P pastoris MXY0291 during submerged fed-batch fermentation and isolated using filtration-based recovery with food- or pharmaceutical-grade materials. The Pichia production strain (MXY0291) is derived from a nontoxigenic and nonpathogenic, well-characterized strain lineage that has a history of safe use in manufacturing proteins for use in food and pharmaceuticals.28,29,32 This process is compliant with the Enzyme Technical Association’s guidelines for fermentation-produced microbiologically derived proteins and follows current Good Manufacturing Practices.33,34 Soy LegH is expressed during submerged fed-batch fermentation. The P pastoris cells in the fermentation broth are lysed by bead mill mechanical shearing. Insoluble material within the lysate is removed by centrifugation and microfiltration. Ultrafiltration is used to concentrate the soy LegH. The resulting concentrated liquid is formulated with sodium chloride and sodium ascorbate and stored as a frozen liquid. Leghemoglobin protein preparation may be stored at −20°C as a frozen liquid for at least 12 months with no observable change in soy leghemoglobin stability or performance in ground beef analogue products. Leghemoglobin protein preparation is a frozen liquid, and the entire final preparation was used for all in vitro safety tests performed in this study. Leghemoglobin protein preparation specifications and batch analysis for the lots used for safety testing are presented in Supplemental Table S1. To aid with homogeneous mixing into the animal diet, LegH Prep was freeze-dried prior to use for all in vivo studies.

The P pastoris LegH production strain MXY0291 was derived from the well-characterized parent strain NRRL Y-11430.31 MXY0291 was modified to overexpress the gene encoding the soy LegH as well as all 8 enzymes in the native Pichia heme biosynthesis pathway (aminolevulinic acid (ALA) synthase, ALA dehydratase, porphobilinogen deaminase, UPG III synthase, uroporphyrinogen (UPG) III decarboxylase, coproporphyrinogen oxidase, protoporphyrinogen oxidase, and ferrochelatase) using the Pichia alcohol oxidase 1 promoter (pAOX1). MXY0291 was also modified to overexpress the Mxr1 transcriptional activator using the pAOX1 promoter. The Mxr1 protein activates the pAOX1 promoter leading to increased expression of pAOX1-driven LegH, heme biosynthesis genes, and Mxr1 itself.

Because there were no test substance-dependent effects observed in the estrous cycle study, a pathology peer review was performed on the initial 28-day dietary feeding study to evaluate distinct estrous cycle stage distribution, decreased presence of fluid-filled uteri and dilated uterine lumens, and decrease in uterine weights observed the group 2 and group 4 females to ensure that these findings were not indicative of a perturbation of the female estrous cycle. The review pathologist evaluated the estrous cycle stage distribution and organ weight and histopathology in all female reproductive organs and corresponding macroscopic and microscopic observations noted by the study pathologist. The decreases in uterine weights, fluid-filled uteri, and dilated uterine lumen did not correlate with any adverse histopathological findings and are therefore interpreted to be nonadverse. The presence of both new and old ovarian corpora lutea in females from all groups indicated that all females were cycling normally. Following the peer review, the study pathologist and review pathologist reached a consensus that there were no test-substance-dependent effects on the female estrous cycle and reproductive organs.

There were no test substance-related changes in average estrus cycle length attributable to LegH Prep administration (). There were no macroscopic or microscopic findings related to the administration of LegH Prep. A single group 2 animal had prolonged estrus based on morphology of the ovaries (large atretic follicles, multiple corpus lutea at a similar state of atresia) and the presence of squamous metaplasia of the uterus. These findings were considered spontaneous and incidental due to the lack of similar findings at higher dose levels. One group 1 animal had large atretic follicles observed in both ovaries, and one group 4 animal had lutenized follicles (follicles with evidence of lutenization in the wall but which have not ovulated) in both ovaries. Both of these observations are reported as background findings in rats of the strain and age used in this study59 and were considered incidental because of their singular occurrences. There were no test substance-related changes in absolute or relative reproductive organ weight values in female rats treated with LegH Prep (). Longitudinal daily monitoring of estrous cycle stage demonstrated that, despite intrinsically normal estrous cycles, the distribution of estrous cycle stages on any given day can be markedly different from the within-rat distribution over time (Supplemental Figure S1).

No mortalities were observed during this study. There were no clinical observations attributable to the administration of LegH Prep. There were no body weight, body weight gain, food consumption, or food efficiency findings considered attributable to LegH Prep administration with the exception of a single incidental increase (P < 0.05) in mean daily body weight, mean food consumption, and mean food efficiency for group 2 animals on days 21 to 28. This increase was transient, nondose dependent, and interpreted to have no toxicological relevance.

A 28-day dietary feeding study was performed with female rats to thoroughly evaluate the estrous cycle stage distributions, decreased presence of fluid-filled uteri and dilated uterine lumens, and decrease in uterine weights observed the group 2 and group 4 females in the previous 28-day dietary feeding study. To ensure all animals had normal estrous cyclicity prior to the 28-day dosing phase, estrous cycle stage was determined daily for all animals for 14 days. Additionally, estrous cycle stage was determined for all animals for the last 14 days of the 28-day dosing period. At study termination, reproductive organs were analyzed. Administered doses of 0 (group 1, control), 512 (group 2, low), 1024 (group 3, medium), and 1536 (group 4, high) mg/kg/d of freeze-dried LegH Prep correspond to 0, 250, 500, and 750 mg/kg/d of LegH, respectively (Supplemental Table S12). The same lot of freeze-dried LegH Prep was used for both 28-day feeding studies. The mean overall daily intake of the test substance in groups 1 to 4 female rats was 0, 250, 496, and 738 mg/kg/d LegH, respectively. The animals are considered to have received acceptable dose levels.

There were no test substance-dependent effects observed during necropsy, organ weights, macroscopic evaluation, and microscopic evaluation in male and female rats, with a single exception of a distinct estrous cycle stage distribution in the female rats (). The estrous cycle consists of 4 stages: proestrus, estrus, metestrus, and diestrous. Each stage has characteristic reproductive organ weights and histopathology. At study termination, groups 2 and 4 females had an increased incidence of animals in metestrus and a decreased incidence of animals in estrus compared to groups 1 and 3. Consistent with the estrous cycle stage distribution, group 2 and 4 females also had decreased presence of fluid-filled uteri and dilated uterine lumens and decreased uterine weights compared to groups 1 and 3 females ( and ). All other microscopic findings at the study day 29/ 30 time point were also unrelated to administration of LegH Prep and can be observed in the age and strain of rats used in this study.57,58 Although the differences in estrous cycle stage distribution between groups was likely due to sampling and assessing estrous cycle distribution on a single day, rather than using a longitudinal study, a more extensive and rigorous longitudinal study was performed focusing on the potential effect of LegH Prep on the estrous cycle.

There were to no test substance-related changes in hematology parameters for male rats (). Statistically significant increase in red BLD cell, hematocrit, and hemoglobin values and absolute basophil counts for group 2 females, and decreased ARETs in group 3 females were nondose dependent and were interpreted to be within the expected biological variation and, therefore, not toxicologically relevant and not test substance dependent (). There were no test substance-related changes in coagulation parameters for female rats. A nondose-dependent increase in APTT was observed in groups 3 and 4 males. Due to its very slight magnitude and lack of correlating pathological or clinical finding, this change is considered nonadverse. There were no test substance-related changes in serum chemistry parameters for male rats (). Alkaline phosphatase was minimally decreased in a nondose-dependent manner for group 2 and group 4 females (). This minimal decrease was not correlated with concurrent clinical pathology or histopathology changes, and due to its limited clinical relevance, it is interpreted to have no toxicological significance and was not test substance dependent. Other differences in serum chemistry parameters that were statistically significant consisted of increased ALB and K values in group 3 males, decreased GLUC and chloride in groups 2 and 3 females, increased GLOB values in group 3 females, and increased CALC in groups 2 and 3 females. These were generally of small magnitude, lacked a response in a dose-dependent manner, and are interpreted to be within expected biological variation and considered to be of no toxicological relevance and non-test substance dependent. There were no test substance-related changes in urinalysis parameters for male or female rats (). Urine sediment analysis was performed for all animals and all results were within normal limits.

No mortalities were observed during this study. There were no clinical observations attributable to the administration of LegH Prep. There were no body weight, body weight gain, food consumption, or food efficiency findings considered attributable to LegH Prep administration (–). A statistically significant decrease (P < 0.01) in mean daily body weight gain was observed in group 2 females on days 14 to 21 (). This decrease was transient and was interpreted to have no toxicological relevance. Statistically significant increases (P < 0.05-0.01) were observed for mean daily food consumption in group 3 males on days 7 to 14 and in group 4 males on days 7 to 10, which were transient and without significant impact on body weight and were interpreted to be nontoxicologically relevant (). Mean food efficiency for the treated female rats in groups 2 to 4 was generally comparable to the control group 1 values throughout the study, with the exception of statistically significant increases (P < 0.01) in group 2 on days 14 to 21 that were transient and without significant impact on body weight and were interpreted to be nontoxicologically relevant (). These small but significant changes were all considered to be nontoxicologically relevant and nontest substance dependent.

A 28-day dietary feeding study in rats was performed to evaluate the potential subchronic toxicity of LegH Prep following continuous exposure of the test substance in the diet. A no observed adverse effect level (NOAEL) was sought for each sex. Due to the successful palatability of 500 mg/kg/d LegH in the previous 14-day feeding study, the maximum dose was increased to 750 mg/kg/d LegH. Administered doses of 0, 512, 1024, and 1536 mg/kg/d of freeze-dried LegH Prep corresponded to 0, 250, 500, and 750 mg/kg/d of LegH, respectively (Supplemental Table S11). The slight difference in correlation between LegH Prep dose levels and LegH concentrations compared to the previous 14-day study are due to the utilization of a different lot of freeze-dried LegH Prep test substance. The mean overall daily intake of the test substance in groups 1 to 4 male rats was 0, 234, 466, and 702 mg/kg/d LegH, respectively. The mean overall daily intake in groups 1 to 4 female rats was 0, 243, 480, and 718 mg/kg/d LegH, respectively. The animals are considered to have received acceptable dose levels.

There were no changes in hematology values attributable to the administration of LegH Prep. There were no LegH Prep-related macroscopic or microscopic findings. Mean absolute and relative organ-to-body weights for groups 2 to 4 were comparable to control group 1 throughout the study. These results suggest that LegH Prep would be well tolerated in a study of longer duration.

There were no mortalities during the course of the 14-day study. There were no clinical observations attributed to the administration of LegH Prep. In-life clinical signs included reported discoloration of the urine in the 6/6 group 4 males and 5/6 group 4 females on day 5. Due to its single-day occurrence and lack of occurrence in any other in-life study, this observation is likely due to rehydration of the test substance by animal urine, which would result in the formation of a red/brown COL. Without a correlation to clinical hematology or any other parameter, these findings are interpreted to be of no toxicological significance. There were no changes in mean food consumption, mean food efficiency, mean body weight, and mean daily body weight gain attributable to the administration of LegH Prep.

A 14-day toxicology and palatability feeding study in rats was performed to assess the feasibility of oral administration of freeze-dried LegH Prep in the diet and to establish the dose range for the subsequent 28-day study. The test substance was administered in doses of 0, 3156, 6312, and 12,612 ppm (groups 1-4, respectively). These concentrations were selected to administer target doses of 0, 263, 525, and 1050 mg/kg/d LegH Prep, corresponding to active ingredient LegH doses of 0, 125, 250, and 500 mg/kg/d, based on the assumption of a 300 g rat consuming 25 g of food per day (Supplemental Table S10). The feed formulation was held constant throughout the study. The calculated nominal dietary intake levels were 0, 134, 269, and 531 mg/kg/d for groups 1 to 4 male rats and 0, 148, 296, and 592 mg/kg/d for groups 1 to 4 female rats, respectively. The animals are considered to have received acceptable dose levels.

In each of the in vivo studies described below, diets were prepared weekly using a food mixer and provided to the animals ad libitum. Each diet preparation was split into half, and 1 aliquot was stored at −20°C. The feed within the animal cages was replaced twice a week, which was a conservative approach implemented to minimize the amount of time that each formulation was held at room temperature. Dietary preparations were analyzed using HPLC to evaluate test substance (freeze-dried LegH Prep) homogeneity and stability and for concentration verification. The neat test substance served as the reference standard for each feed measurement. In each case, the analyte fell within acceptable parameters: <10% relative standard deviation of LegH Prep concentration between samples of the feed collected from top, middle, and bottom of the mixer, indicating homogeneous distribution; <10% change in LegH Prep concentration during diet presentation, indicating stability; and within 10% of the target concentration of LegH Prep, indicating accurate dosing. Detailed analytical chemistry HPLC results from the 28-Day Dietary Feeding Study in Rats are presented in Supplemental Tables S6 to S9.

The increased incubation time of experiment 2 resulted in precipitation of the test substance at concentrations of ≥500 µg/mL LegH. Additionally, the mitotic index values relative to the control decreased below the 45% threshold at concentrations >1000 μg/mL LegH. Therefore, only concentrations up to 1000 μg/mL LegH were evaluated for chromosome aberrations. The proliferation index values for 500 and 1000 µg/plate were 1.23 (79% relative to control) and 1.12 (72% relative to control), respectively (). This decrease was not a consequence of chromosome aberrations. In experiment 2, no significant increase in cells with structural or numerical chromosome aberrations was observed up to 1000 μg/mL LegH, which was the maximum dose evaluated, without metabolic activation ().

In accordance with OECD guidelines, experiment 1 used the recommended concentrations of 100 to 5000 μg/mL LegH, which corresponded to 148 to 74 000 μg/mL LegH Prep (Supplemental Table S5). Mitotic index was evaluated first, since decreased mitotic index can inhibit the ability to evaluate chromosome aberrations. Although the mitotic index decreased to below 70% of the negative control at high concentrations of LegH Prep without S9 metabolic activation, no such decrease in the mitotic index was observed in the presence of S9 metabolic activation (). No significant difference in the proliferation index was observed under either condition (). Evaluation of chromosomal aberration tests using the mitotic index can be unreliable.56 However, in all cases, the mitotic index remained above the 45% of control threshold that is recommended for evaluation of structural and numerical chromosomal aberrations, and no test substance precipitation was observed. In experiment 1, no significant increase in cells with structural or numerical chromosome aberrations was observed up to 5000 μg/mL LegH, which was the maximum dose tested, both with and without S9 ().

The objective of this in vitro assay was to evaluate the ability of LegH Prep to induce structural or numerical (polyploid or endoreduplicated) chromosome aberrations in HPBL. Human peripheral BLD lymphocytes cells were exposed to LegH Prep for 4 hours in the presence or absence of S9 (Experiment 1) or for 24 hours in the absence of S9 (Experiment 2). In each experiment, untreated and positive control values were within the historical control range indicating that the subject assay met the criteria for a valid test (Supplemental Table S4a-c).

Leghemoglobin protein preparation did not cause a positive increase in the mean number of revertant colonies per plate with strains TA1535, TA1537, TA98, TA100, or WP2 uvrA in either the absence or the presence of S9 when using either the plate incorporation or the preincubation method (). Therefore, LegH Prep was nonmutagenic in the bacterial reverse mutation assay.

The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, considering the laboratory historical control range and/or published values (Supplemental Table S3).54,55 The positive control substances caused the expected substantial increases in revertant colony counts in both the absence and the presence of S9 in each phase of the test, confirming the sensitivity of the test and the activity of the S9 mix (, Supplemental Table S3). No signs of precipitation or contamination were noted throughout the study. Therefore, each phase of the test is considered valid.

The objective of this test was to determine the mutagenic potential of LegH Prep using histidine-requiring strains of S. typhimurium (TA98, TA100, TA1535, and TA1537) and a tryptophan-requiring strain of E. coli (WP2 uvrA). Leghemoglobin protein preparation was evaluated with and without an exogenous metabolic activation (S9 mix) at levels of 23.384, 74, 233.84, 740, 2338.4, 7400, 23,384 and 74,000 μg/plate, which corresponded to 1.58, 5.0, 15.8, 50, 158, 500, 1580, and 5000 μg/plate of the characterizing component, LegH, with the high level being the standard limit for this test (Supplemental Table S2).

Discussion

Heme is ubiquitous in the human diet and has been consumed for thousands of years. Replacing the myoglobin that catalyzes the unique flavor chemistry of meat derived from animals with LegH from soy opens an opportunity to develop plant-based meats that deliver to consumers the pleasure they demand from animal-derived meats, with a small fraction of the environmental impact. Leghemoglobin protein preparation is manufactured using a P pastoris production strain that has been engineered to overexpress LegH. Following submerged fed-batch fermentation, the Pichia cells are lysed and the LegH is isolated using a filtration-based recovery process. The LegH Prep ingredient contains 6% to 9% LegH, which makes up at least 65% of the total protein fraction. The balance of the proteins is from the Pichia host. Leghemoglobin protein preparation is stabilized with NaCl and sodium ascorbate. A complete analysis of the LegH Prep specifications and chemical composition is provided in Supplemental Table S1.

A previous study evaluating the safety of LegH Prep in food used in silico approaches, such as literature searches for reports of allergenicity or toxicity and extensive sequence homology comparisons to databases of known allergens and toxins.30 That study also analyzed sensitivity to pepsin digestion in an in vitro simulated gastric fluid. Collectively, the previous work concluded that food products containing LegH Prep posed a low risk of allergenicity and toxicity to consumers.30 In this study, we evaluated the safety profile of LegH Prep through a series of in vitro and in vivo studies.

The Pichia-derived LegH Prep was nonmutagenic in the bacterial reverse mutation test, which evaluated 5 strains of bacteria and 8 different concentrations of LegH Prep up to a maximum dose of 5000 μg LegH/plate. Similarly, LegH Prep was nonclastogenic in the chromosomal aberration test, which evaluated chromosomal rearrangements in HPBL following 4-hour (with and without metabolic activation) and 24-hour (without metabolic activation) incubations with LegH Prep. These assays tested LegH concentrations up to 5000 μg/mL for the 4-hour incubations. Due to test substance precipitation and decreased percent mitotic index, 1000 μg/mL LegH was the maximum dose evaluated for the 24-hour incubation. Together, these results demonstrate that LegH Prep is nonmutagenic and nonclastogenic under the in vitro conditions tested.

To evaluate the in vivo safety profile for potential systemic toxicity, a 28-day feeding study was conducted in rats in which LegH Prep was administered in the diet. There were no LegH Prep-dependent effects observed with the exception of a distinct estrous cycle stage distribution in groups 2 and 4 females at study termination. Groups 2 and 4 females had an increased incidence of the metestrus stage of the estrous cycle (), decreased presence of fluid filled uteri and dilated uterine lumens, and decreased uterine weight compared to groups 1 and 3 females ( and ). However, the correlation between estrous cycle stage and reproductive organ weight and pathology for each animal was consistent with published literature on normal healthy rats.60 Therefore, although the estrous cycle stage distribution was different between the groups, there were no data to suggest an adverse impact on the health of the female animals; the presence of both new and old ovarian corpora lutea indicated normal estrous cyclicity.59 Without evidence of an adverse effect in the female ovary or uterus pathology, the decrease in relative and absolute uterine weights in groups 2 and 4 females was interpreted to be nonadverse. Moreover, decreased uterine weight is normal for animals in the metestrus stage of the estrous cycle.61

To thoroughly evaluate the estrous cycle stage distributions, decreased presence of fluid-filled uteri and dilated uterine lumens, and decrease in uterine weights observed the group 2 and group 4 females, an in-depth follow-up 28-day dietary feeding study in female rats was performed with longitudinal estrous cycle monitoring and evaluation of reproductive organ weights, gross necropsy, and histopathology. The results demonstrated that LegH Prep had no impact on the estrous cycle length, distribution, or female reproductive organ health when administered at dose levels up to 750 mg/kg/d LegH for 28 days ( and ). Despite intrinsically normal estrous cycles, different estrous cycle stage distributions were observed between groups on any given day (Supplemental Figure S1). This highlights the importance of longitudinal estrous cycle monitoring to evaluate estrous cyclicity. For example, if the estrous cycle stages were only monitored on a single day, a completely different conclusion would have been drawn regarding the test substance effect on estrous cycle if the animals had been analyzed, for example, on day 18 of the dosing period compared to day 21 (Supplemental Figure S1). The single-day sampling artifact readily accounts for the increased incidence of metestrus observed in the initial 28-day dietary feeding study. Moreover, a pathology peer review of the original 28-day study resulted in a consensus between the study pathologist and review pathologist that LegH Prep did not affect the estrous cycle. This is consistent with the lack of any scientific literature identifying phytoestrogens in Pichia as well as the absence of any published incidence of heme proteins affecting the estrous cycle.

The intake of LegH was assessed as the EDI and a 90th percentile user, representing the worst-case scenario in the consumption of LegH as a flavor catalyst in meat replacement products. Safety was assessed as a function of the actual exposure, and feeding study dose levels were chosen purposefully to reflect the actual exposure situations that would be encountered by a user. LegH Prep will not be sold to consumers as an individual ingredient and will instead be included in plant-based meat products at a level not exceeding 0.8% LegH. Together, these systemic toxicity and reproductive health feeding studies in rats established an NOAEL of 750 mg/kg/d LegH for both sexes, which was the maximum dose administered. The acceptable daily intake (ADI) is calculated by dividing the NOAEL by an acceptable uncertainty factor. In the absence of extenuating circumstances, the food additive regulations 21 C.F.R. 170.22 recommend a factor of 100. The ADI for soy leghemoglobin is 750/100 or 7.5 mg/kg/d. The 90th percentile EDI for soy leghemoglobin is 6.67 mg/kg/d. Based on FDA guidelines, since the EDI is lower than the ADI, these results suggest there are no safety concerns.62 Collectively, these in vitro and in vivo results suggest that LegH Prep, containing both soy LegH and Pichia proteins from the production host, raise no issues of toxicological concern under the conditions tested.

Creating safe, delicious plant-based meats to replace animal-derived meats in the diet is critical to reducing and eventually eliminating the environmental impact of the animal farming industry. Impossible Foods Inc has shown that plant-based meat containing up to 0.8% LegH delivers flavors and aromas that are characteristic of animal-derived meat.11 This study established an NOAEL of 750 mg/kg/d LegH, which is over 100 times higher than the 90th percentile EDI. This maximum dose is equivalent to an average-sized person (60 kg) consuming 5625 g (12 lbs) of plant-based ground beef analogue with 0.8% LegH per day. Thus, the results of the studies presented in this article raise no questions of toxicological concern under the conditions tested for LegH Prep, which is intended for use in ground beef analogue products at levels up to 0.8% LegH.


Ames, IA - Profile data - Census Reporter

Per capita income. about 90 percent of the amount in the Ames, IA Metro Area: $32,402. ±$2,460. about 80 percent of the amount in Iowa: $33,107. ±$451. $50,528. ±$11,064. Median household income. about 80 percent of the amount in the Ames, IA Metro Area: $62,181 †.Census data for Ames, IA (pop. 66,258), including age, race, sex, income, poverty, marital status, education and more..
From: censusreporter.org


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image of AMES INSTRUMENTS CM600A 600A AC Clamp Meter – Item …

AMES INSTRUMENTS CM600A 600A AC Clamp Meter – Item …

The AMES™ INSTRUMENTS 600A AC Clamp Meter provides a full range of AC current, AC/DC voltage, and frequency measurement functions for basic electrical troubleshooting. The AC clamp meter has a 3-3/4 digit digital display capable of measuring resistance, continuity and diode test. The unit also has a built-in work light and non-contact voltage measurement capability…The AMES™ INSTRUMENTS  600A AC Clamp Meter provides a full range of AC current, AC/DC voltage, and frequency measurement functions for basic electrical troubleshooting. The AC clamp meter has a 3-3/4  digit digital display capable of measuring resistance, continuity and diode test.  The unit also has a built-in work light and non-contact voltage measurement capability….
From: go.harborfreight.com


Ames® - Our Tools Built America - YouTube

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From: www.youtube.com


エームズ試験 - Wikipedia

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From: ja.wikipedia.org


CM400A 400A AC Clamp Meter - Harbor Freight Tools

The Ames 400A AC clamp meter features an ergonomic design which greatly increases comfort and ease-of-use in a variety of applications. Some of its main features include AC current measurement, worklight, and a multi segment 4000 count display with backlight. This electrical tester is designed for professional residential and commercial ....
From: www.harborfreight.com


600A, AC/DC TRMS Clamp Meter (CL2000) Instruction Manual

139705 Rev. 09/14 B SERVICIO AL CLIENTE KLEIN TOOLS, INC. 450 Bond Street Lincolnshire, IL 60069, EE. UU. 1-877-775-5346 [email protected]
From: data.kleintools.com


Genetics Ch. 18 HW Flashcards | Quizlet

The Ames test determines the frequency with which a chemical causes mutations in DNA. The results of the Ames test for the substances X, Y, and Z are provided. Label the carcinogenic potential of each of these substances based on the production of his+ revertants in the presence or absence of liver extract (± LE)..
From: quizlet.com


Ames, IA Metro Area - Profile data - Census Reporter

Per capita income. about the same as the amount in Iowa: $33,107. ±$451. about 90 percent of the amount in United States: $35,672. ±$76. $62,181. ±$6,607. Median household income. about the same as the amount in Iowa: $61,691.Census data for Ames, IA Metro Area (pop. 123,351), including age, race, sex, income, poverty, marital status, education and more..
From: censusreporter.org


High Fidelity Simulation at NASA Ames Research Center

High Fidelity Aerospace Simulations at NASA Ames SimLabs. Mike Jackson, Srba Jovic, Peter Zaal Presented by: Mike Jackson, Srba Jovic. 1.
From: ntrs.nasa.gov


Bruce Edwards (Lynn), 50 - Ames, IA Public Reputation ...

Bruce Edwards's birthday is 07/23/1971 and is 50 years old. Previously city included Avoca IA. Bruce Lynn Edwards, Bruce A Edwards, Bruce L Edwards and Bruce Staci Edwards are some of the alias or nicknames that Bruce has used. Background details that you might want to know about Bruce include: ethnicity is unknown, whose political affiliation ....
From: www.mylife.com


High Fidelity Aerospace Simulations at NASA Ames SimLabs

Vertical Motion Simulator - 40 Years of Innovation 1980 1990 2020 2000 2010 XV-15 Crew Exp. Vehicle Lunar Surf. Acc. Module X-32B Space Shuttle USAir 427.
From: ntrs.nasa.gov


LA-LT-041 Lexington Ames SJSA Single Jacket Light Armored ...

The Lexington Ames All Dry LA-LT-041 series is ideal for FTTH projects where cables are direct buried or installed in a conduit. The LA-LT-041, Single Mode, 9/125, G.652.D is manufactured with GR-20 certified glass. Our estimated lead time for this product is approximately 16 weeks, subject to global transportation delays • No Tariffs • Shorter Lead Time • Great Prices • Save ….
From: www.balticnetworks.com


image of Ames, TX | Data USA

Ames, TX | Data USA

In 2019, Ames, TX had a population of 1.39k people with a median age of 38.6 and a median household income of $26,179. Between 2018 and 2019 the population of Ames, TX grew from 1,247 to 1,394, a 11.8% increase and its median household income grew from $24,176 to $26,179, a 8.29% increase.In 2019, Ames, TX had a population of 1.39k people with a median age of 38.6 and a median household income of $26,179. Between 2018 and 2019 the population of Ames, TX grew from 1,247 to 1,394, a 11.8% increase and its median household income grew from $24,176 to $26,179, a 8.29% increase..
From: datausa.io


image of Machine learning – Predicting Ames mutagenicity of small ...

Machine learning – Predicting Ames mutagenicity of small ...

Dec 01, 2021 · 1. Introduction. Within the modern drug discovery field, the mutagenicity of a compound is a crucial property that can restrict the development of a particular compound series at all stages of drug development due to its close relationship with carcinogenicity [1,2].In order to assist the early identification of potential mutagenic compounds and hence reduce the time …In modern drug discovery, detection of a compound's potential mutagenicity is crucial. However, the traditional method of mutagenicity detection using….
From: www.sciencedirect.com


Owner’s Manual & Safety Instructions

Item 64014 For technical questions, please call 1-888-866-5797. Page 7 Sa FE ty Op E rati O n Maint E nanc E S E tup Display Symbol Description autO Auto range mode Data Indicates that display data is being held Diode test Continuity buzzer Battery low indicator Dc Direct current ac Alternating current °c °F Celsius / Fahrenheit (temperature) kMΩ Ohms, Kilohms, Megohms ….
From: manuals.harborfreight.com


image of Ames v. Home Depot U.S.A., Inc., No. 1:2008cv06060 ...

Ames v. Home Depot U.S.A., Inc., No. 1:2008cv06060 ...

Ames v. Home Depot U.S.A., Inc., No. 1:2008cv06060 - Document 48 (N.D. Ill. 2009) Court Description: MEMORANDUM Opinion and Order Signed by the Honorable David H. Coar on 12/2/2009:Mailed notice (pm, ) Download PDF. IN THE UNITED STATES DISTRICT COURT FOR THE NORTHERN DISTRICT OF ILLINOIS EASTERN DIVISION DIANE AMES, ) ) ) ) ) …Ames v. Home Depot U.S.A., Inc., No. 1:2008cv06060 - Document 48 (N.D. Ill. 2009) case opinion from the Northern District of Illinois U.S. Federal District Court.
From: law.justia.com


image of Early life growth hormone treatment shortens longevity and ...

Early life growth hormone treatment shortens longevity and ...

Hypopituitary Ames dwarf mice were injected either with growth hormone (GH) or thyroxine for a 6-wk period to see whether this intervention would reverse their long life span or the resistance of their cells to lethal stresses. Ames dwarf mice survived 987 ± 24 d (median), longer than nonmutant cont …Hypopituitary Ames dwarf mice were injected either with growth hormone (GH) or thyroxine for a 6-wk period to see whether this intervention would reverse their long life span or the resistance of their cells to lethal stresses. Ames dwarf mice survived 987 ± 24 d (median), longer than nonmutant cont ….
Keyword: pmid:20720157, PMC2992365, doi:10.1096/fj.10-163253, Research Support, N.I.H., Extramural, Jacob A Panici, James M Harper, Michal M Masternak, Animals, Body Weight / drug effects, Cell Survival / drug effects, Cells, Cultured, Dwarfism, Fibroblasts / cytology, Fibroblasts / drug effects, Growth Hormone / pharmacology*, Longevity / drug effects*, Male, Mice, Thyroxine / pharmacology, PubMed Abstract, NIH, NLM, NCBI, National Institutes of Health, National Center for Biotechnology Information, National Library of Medicine, MEDLINE
From: pubmed.ncbi.nlm.nih.gov


Owner’s Manual & Safety Instructions

Item 64015 For technical questions, please call 1-888-866-5797. Page 5 Sa F ety Operati O n Maintenance Setup Specifications DC Voltage Ranges: 660mV / 6 6V / 66V / 600V DC Voltage Accuracy (@660mV to 66V) ± 0 8%of rdg + 3D.
From: manuals.harborfreight.com


Quand se révèlent les âmes selon Patrick Cothias ...

Quand se révèlent les âmes selon Patrick Cothias - Citation du 30.12.2021. Publié depuis 12 jour (s) le jeudi 30 décembre 2021 - Lien permanent ±. Nombre d'évaluation : 29 - Note : 4.17. C'est dans la douleur et l'adversité que les âmes se révèlent....
From: www.dicocitations.com


image of Validating a refractometer to evaluate immunoglobulin G ...

Validating a refractometer to evaluate immunoglobulin G ...

The objectives of this study were to (1) validate a method using refractometry to rapidly and accurately determine immunoglobulin (IgG) concentration in Jersey colostrum, (2) determine whether there should be different refractive index (nD) and %Brix cut points for Jersey colostrum, and (3) evaluate …The objectives of this study were to (1) validate a method using refractometry to rapidly and accurately determine immunoglobulin (IgG) concentration in Jersey colostrum, (2) determine whether there should be different refractive index (nD) and %Brix cut points for Jersey colostrum, and (3) evaluate ….
Keyword: pmid:25465569, doi:10.3168/jds.2014-8730, Research Support, Non-U.S. Gov't, Validation Study, K M Morrill, K E Robertson, H D Tyler, Animals, Cattle / metabolism*, Colostrum / chemistry*, Female, Freezing*, Immunodiffusion / veterinary*, Immunoglobulin G / analysis*, Refractometry / instrumentation, Refractometry / veterinary*, PubMed Abstract, NIH, NLM, NCBI, National Institutes of Health, National Center for Biotechnology Information, National Library of Medicine, MEDLINE
From: pubmed.ncbi.nlm.nih.gov


image of Prevention of neuromusculoskeletal frailty in slow-aging ...

Prevention of neuromusculoskeletal frailty in slow-aging ...

Caloric restriction (CR) has similar effects on healthspan and lifespan, and causes an extension of longevity in Ames dwarf mice. Our study objective was to determine whether Ames dwarfism or CR influence neuromusculoskeletal function in middle …Ames dwarf (Prop1 (df/df) ) mice are remarkably long-lived and exhibit many characteristics of delayed aging and extended healthspan. Caloric restriction (CR) has similar effects on healthspan and lifespan, and causes an extension of longevity in Ames dwarf mice. Our study objective was to determine ….
Keyword: pmid:24155868, PMC3796515, doi:10.1371/journal.pone.0072255, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Oge Arum, Zachary Andrew Rasche, Andrzej Bartke, Aging / physiology*, Animals, Body Weight, Caloric Restriction*, Dwarfism / genetics*, Female, Hand Strength / physiology, Humans, Longevity / genetics*, Male, Mice, Mice, Mutant Strains, Motor Activity / physiology, Musculoskeletal System / innervation*, Physical Fitness / physiology*, PubMed Abstract, NIH, NLM, NCBI, National Institutes of Health, National Center for Biotechnology Information, National Library of Medicine, MEDLINE
From: pubmed.ncbi.nlm.nih.gov


COMPARATIVE MUTAGENICITY OF ETHANOL, ACETONE AND ...

The Salmonella Ames assay is one of the most widely used short-term basic regulatory tests to assess the mutagenic potential of new ... Mean ± SD 133.33 ± 3.79 130.33 ± 15.63 133.00 ± 13.86 127.67 ± 4.51 140.33 ± 18.01 TA98 Individual Plate Count (n=3) 17 17 21 17 25 18 23 12 19 29 21 23 25 27 21 ....
From: innovareacademics.in