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Pressure BioSciences, Inc.Pressure Cycling Technology (PCT):Advancements inBiological Sample PreparationPotentialApplications in Forensic ScienceDiscovery Starts With Sample Preparation

History of High Pressure in Life Sciences 1623-1662: Blaise Pascal – described fundamentalconcepts of pressure and vacuum 1895: H. Royer – pressure kills bacteria 1899: B.H. Hite et al. – pressure preserves milk 1914: P.W. Bridgman - pressure coagulates egg white 1989: High pressure processing of food products 2000: First International Conference on HPBB 2008: Fifth International Conference on HPBB in the USA

Pressure Cycling Technology (PCT)PCT is a Novel, Enabling Technology thatUses Cycles of Hydrostatic Pressure BetweenAtmospheric and Ultra-high Levels (up to35,000 psi and greater) to Allow for thePrecise Control of Biomolecular Interactions

Understanding Hydrostatic PressureU.S. Navy BathyscapheTrieste (1958-1963)Marianas Trench:38,713 ft (11,800m) deep16,000 PSI (120MPa)Significant portion of the Global Biosphere issubjected to high hydrostatic pressure!

Pressure: a Thermodynamic Process Pressure is a measurement of the force exerted per unitarea on the boundaries of a substance or system. It is caused by the collisions of the molecules of thesubstance with the boundaries of the system. As molecules hit the walls, they exert forces that try topush the walls outward. The forces resulting from all these collisions cause thepressure exerted by a system on its surroundings.

Compressibility Organic material is more compressible than water PCT exploits the differences between compressibilityof different components of the sample Compression heating is proportional tocompressibility Hydrostatic pressure does not cause shearing

Synergy of Physics and Chemistry PCT selectively disrupts membrane structures based ontheir size and fluidity PCT is compatible with a wide variety of reagents Enzymatic lysis is enhanced by Pressure Cycling Temperature and pressure are generally synergistic PCT can be combined with affinity purification

Current Extraction Methods Mortar & PestleDounce homogenizer (glass on glass)Potter-Elvenhjem homogenizer (Teflon on glass)Enzymatic DigestionPolytron shearing homogenizersBlendersBead BeatingSonicationRepeated Freeze/Thaw cyclesFrench Press ( 2000 PSI)

State of the Art?“A collaborator at a major university in a multi-milliondollar Proteomics Facility, equipped with the mostadvanced instrumentation but they use mortar and pestle.”Native American IndianMortar and Pestlecirca 1000 AD

PCT Sample Preparation SystemHydraulic System3 Samples SimultaneouslyOptional Temperature ControlBarocyclerTM NEP3229

PCT Sample Preparation SystemPneumatic SystemSingle Sample CapacityOptional Temperature ControlTMBarocyclerNEP2320

User-Adjustable Variables Pressure (up to 35 kpsi) Number of Cycles Cycle Profile Chemistry Temperature

The FT500 PULSE TubeSpecially designed multi-functional tube Single-UseVersatile, works with:- Standard and custom reagents- Various sample types- Range of sample sizesConvenientEfficientSafe: closed tube, sample fully-contained

The PULSE Tube

Diskless PULSE TubeFT500-ND PULSE Tubes

Variable Volume PULSE Tube FT 500-ND100 – 1500 uL Cell Suspensions Emulsions Soil samples In-solution digestionSample chamberCapRam

MechanicalThe PCT Shredder

PCT MicroTube

PCT MicroCaps

ID in 60 Minutes

Forensic Programs Evaluating PCTUniversity of North TexasLow Copy Number (Touch Samples, Swabs)Marginal Quality BoneMitoDNA from HairDr. Bruce BudowleMinnesota Department of Public SafetyMarginal Quality BoneLow Copy Number (Touch Samples, Swabs)Dr. Anne GrossFlorida International University (1)Differential Lysis of Sperm and Vaginal CellsDr. Bruce McCordFlorida International University (2)Vacuum FiltratesDr. Dee MillsPressure BioSciences, Inc.Low Copy Number (Touch Samples, Swabs)Dr. Alexander LazarevDr. Vera GrossPBI is extremely grateful to Promega Corp.and Mr. Len Goren (Global Director, Genetic Identity Promega Corp.)for supporting the development of novel PCT-enhancedforensic methods in these laboratories.

In SightAgain, the front-end sample preparationis perhaps the biggest hurdle toovercome, since crime-samples presentthemselves in myriad manners and thesemacro-samples may not be readilyamenable to microfluidic processing.Bruce Budowle and Angela van Daal

mtDNA from a Single Human HairPCTNon-PCT-PCR

Hair SamplesAverage mtDNA PCR Product Yield of Extraction MethodsAverage mtDNA PCR Product (ng/uL)0.90.80.70.60.50.40.30.20.10Organic with PCTOrganic without PCTDNA IQ with PCTExtraction MethodDNA IQ without PCT

mtDNA from Human Skin Collected on TapePCTNon-PCTPCR-

mtDNA from Human Blood from a Single FiberPCTNon-PCT PCR

Genomic DNA from Pig Bone

Bone #10: No Pressure

Bone #10: with PCT

Bone #4: No Pressure

Bone #4: with PCT

Bone #1No PressurePCT

Average DNA Yields from Bone

DNA Extraction from Human Bone Samples (Korean Samples)Gel One: PCR products of mitochondrial geneGel two: PCR products of human B-actin 45678910

Differential Lysis of Sperm and Vaginal CellsDifferential Lysis: Exploiting The Pressure ProfileVariables Pressure Number of Cycles Buffer Temperature

“Live” Kidney Mitochondria Isolated by PCT

SummaryThe PCT SPS combines the elements of most competingsample preparation technologies in one powerful package: Compatible with the chemical disruption buffers and downstream analysisbuffers Little or no grinding and shearing that destroy macromolecules Precise temperature control, including cryogenic conditions Uniform energy distribution throughout the sample Precise control of molecular disassembly Compatible with a wide variety of organic solvents No exposure to metal parts – preserves fragile redox-active chemical speciesand prevents activation of metalloproteases

1895: H. Royer – pressure kills bacteria 1899: B.H. Hite et al . – pressure preserves milk 1914: P.W. Bridgman - pressure coagulates egg white 1989: High pressure processing of food products 2000: First International Conference on HPBB