6. de long - natural materials

8/7/2019 6. De Long - Natural Materials

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Natural Materials & SystemsMarch 2011

Dr. Hugh C. De LongInterim Director

AFOSR/RSLAir Force Office of Scientific Research

AFOSR

Distribution A: Approved for public release; distribution is unlimited. 88ABW-2011-0778


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Name: Hugh C. De LongPortfolio Description:

The goals of this program are to: 1) study, use, mimic, or alter howbiological systems accomplish a desired (from our point of view) task, and2) enable them to task-specifically produce natural materials and systems.

Both goals are to advance or create future USAF technologies.

List Portfolio Sub-Areas*:

2306 DX Biomimetics2306 DX Natural Materials

2306 DX Natural/Synthetic Interfaces2306 EX Extremophiles

*All four sub-areas include

international programs

2011 AFOSR SPRING REVIEW2306 DX PORTFOLIO OVERVIEW


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Scientific Challenges

Bio-camouflage To achieve rapid adaptive coloration.

Payoff: Would make current camouflage ideas obsolete.

Peptide mediated materials synthesis Looking to learn how materialseither bind to or grow on peptides.

Payoff: Get to a predictive state for sequence or material.

Structural Coloration Identifying the role of hierarchical self-assembly

Payoff: Create new empirically supported paradigm of developmentof highly ordered biophotonic nanostructures.

Cell-Directed Assembly Using cells to build nanostructuredarchitectures with engineered bio/nano interfaces

Payoff: Understand cellular behavior and allow understanding ofbiology at individual cell level

Chromophores To discover or engineer new chromophores in desiredwavelength regions

Payoff: New materials in desired wavelength regions


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Transformational Opportunities

Bio-Camouflage (cephalopods sole animal group with controllable skin

texture) important to learn how and when they are deployed in changingbackgrounds

will transform approaches to mission coats and enable an efficient(passive) mechanism to regulate brightness & contrast

Rational design of peptides for material synthesis and assembly engineered

material and material systems

leads to new method to fabricate sensors; other multifunctional materials

Soft Lithography - a unique approach to synthesize and position single sub-10-nm nanoparticles

create nanostructures small enough to behave as single particle devices(e.g., sensors) integrated with prefabricated circuitry.

Structural Coloration will look to understand the biological strategiesinvolved in the manipulation of light, and structural color

it will produce new processing technologies to fabricate bio-inspired

tunable optical architectures with optimal structure and properties atmultiple length scales.


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Other Organizations That FundRelated Work

Chromophores I currently have two grants plus work in AFRL. The work of

other organizations is almost exclusively on reporter technology. The interestof the AFOSR program is on wavelength, intensity, and lifetime as it pertainsto marking items.

Silk DARPA has contributed to my existing program. ARO has a singlegrantee. NSF has several single PI grants.

Structural Coloration/Bio-Camouflage MURI with ONR focused on visionaspect. ARO has a single grant with ICB PI. NSF has just single PI grants.

Biomolecular assembly A number of funding organizations are interested inthis area, so the AFOSR program is focused on soft lithography, peptidebinding, and self or directed assembly for materials. AFRL program worksclosely with this group for both relevance and guidance.

Extremophiles NASA has funded this area and focused on the origins of life.The focus of the AFOSR program is on radiation protection mechanisms, bio-templating, and biopolymers that can exist in extreme environments. ARO isfocused on spore formers.


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Program Trends

Chromophores/Bioluminescence Bio-X STT phase 1 focus. One of

its discoveries are now used by AFRL TDs, Navy & several Univ PIs Bio-camouflage New PBD 709 program for FY09 looking at

iridiphores, leucophores, chromatophores, papillae, & control system.This program is linked to a new program in AFRL/RX in FY2011.

Structural Coloration new area, several PIs & new MURI (Harvard)

Biopolymers Mainly silk but looking at other biopolymers. The silkwork is well integrated with AFRL; many exchanges of personnel &material.

Biomolecular assembly New MURI at Georgia Tech, rest hasremained constant.

Peptide Mediated Materials Synthesis The efforts are focused ondiscovering the nature of the mechanism behind this.

Extremophile survival Focus shifted to materials within cells thatwork under these conditions. Looking at mechanisms of proteinactivity under extreme conditions with the goal to transfer good ideas

into weaker systems. Fewer PIs left that perform this type of work.


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Recent Transitions

Endogenous Green Fluorescent Protein (GFP) from Amphioxus (UCSD) transitioned toAFRL/RX & RH as well as Navy and other University PIs

Thin film flexible device with unique optical properties for military applications-multi-laminated structure to mimic cephalopod skin (AFRL/RX) transitioned (AFRL & Army 6.2)

Encapsulation of enzymes in silk films and fibers for decon applications (AFRL/RX)(transition to DTRA program)

Peptide Functionalized Nanoparticles for Environmental Sensing (AFRL/RX) UES, Inc.& Woolpert, Inc

Intracellular detection and tagging demonstrated with Aptamer Coated Nanoparticlesthat release reporter flares (NWU) single base-pair mismatch selectivity licensed toAurasense, Inc

Pretreatment using IL on Corn Stover (NCSU) transitioned to Novozyme as only system

that will allow them to commercially process corn as a cellulosic biofuel. Tetratopic Phenyl Compounds, Related Metal-Organic Framework Materials, (NWU).

Product is currently commercialized by Sigma-Aldrich Corp.

Silk-electronic interfaces for disposable optical transmitters (Tufts U) transitioned tothe DARPA chemical communications program

Use of low light digital spectrograph analysis on biomaterials (UCSD) - Ken Browne(GenProbe)


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StatusQu

o

NewInsights

&Approaches

Main AchievementsQuantitativeImpact

EndGoals

200 m

Significantly advanced chemicaltoolkit and theoretical knowledge forthe construction of abioticsupramolecular enzyme mimics.

Created abiotic systems which canallosterically catalyzepolymerization, self-amplify smallmolecule substrate, selectively

recognize Cu(II) ions, control theorientation of multiple catalyticporphyrin sites in MOFs and radicalpairs in mechanostereochemicalassemblies, and selectively bindsmall molecule guests in highlyporous materials.

Established theoretical foundationfor studying catalytic porphyrinMOFs in solution.

Materials for small molecule

separations and storage.

Selective sensors for chemicaland biological targets.

High density information storage.

Compact energy generation,conversion and storage materials.

No versatile assembly methods

for radicals and highly reactivespecies are available.

Detection schemes for non-biological self-amplification arenon-existent .

Allosterism in supramolecularsystems is not well-established.

Radically-enhanced molecularrecognition properties enableaccess to stable radical dimers inthe mechanically interlockedassemblies.

Rational design of supramolecularbuilding blocks and the associatedassembly methods can provide aplatform to switchable catalysts.

Elucidation of catalytic mechanismfor enhanced rates in MOFs viaadvanced Grand Canonical MonteCarlo simulations.

Supramolecular assemblytechnique can be implemented forsynthesis of enzyme mimics,which delineate the principles forrecognition and catalysis.

First proof-of-principle

demonstration of artificial PCR. A series of structurallyunprecedented porphyrin-basedMOF catalysts was developed.

Learned how to control poreactivation and catenation inMOFs.

First allosteric single-site catalystwas developed.

Bio-inspired Supramolecular Enzymatic SystemsPI: C. A. Mirkin; Co-PIs: J. T. Hupp, S. T. Nguyen, J. F.

Stoddart, M. A. Ratner

All i S i h S h i d


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Allosteric Switch Synthesis andConcept


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Artificial PCR via WLA


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Created and studied the first artificial mimic of PCR.

Designed and synthesized a triple-decker allosteric supramolecularcatalyst which can catalyze an allosterically controlled polymerizationreaction.

Developing general routes towards controlled allosteric regulation with

complex substrates via relevant coordination chemistry. Currently investigating multiple heteroligated structures which can act

as allosteric enzyme mimics in the context of substrate recognition,amplified detection and catalytic bond activation.

Colorimetric detection of Cu(II) ions using DNA modified goldnanoparticles has been accomplished, new detection scheme usinginfinite coordination polymer nanoparticles is currently pursued.

Summary


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high r.i.

light

low r.i.

Multilayer Reflectors

(Vukusic)

Structural coloration is common in nature (insect wings, bird feathers,

fish scales, cephalopods, cat eyes) Accomplished through diffraction, interference, and differences inrefractive indices (most common form is the multilayer reflector) Natures design can be used to inspire a new generation of (dynamic)optical materials and devices

Structural Coloration


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5 m

Chromatophores(Oregon State U)

Iridophores(AFRL)

Leucophores(Woods Hole)

J. B. Messenger (2001) Biol. Rev. 76473-

528.

4 m

SEM Micrograph by Hanlon &Mathger

5 m

Cephalopods: Multi-Layered Structureuses both structural

and pigmentcoloration

D t i ti f th Mi i l F ti l U it f
http://www.mbl.edu/inside/what/news/press_releases/documents/RSBL20060542.pdfhttp://www.mbl.edu/inside/what/news/press_releases/documents/RSBL20060542.pdf


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Using a subset of sequences available, have already

begun to identify residues that are highly conservedamongst reflectins.

Gathered evidence that reflectins are present outsideof cephalopods, in Tridacna, the giant clam.

Cloned and began expression of reflectin constructsto examine impact of repeat number on reflectinactivity. Assays showing that only 2 repeats may benecessary for function.

Determination of the Minimal Functional Units ofReflectins

Wendy Crookes-Goodson and Rajesh Naik, AFRL/RXBN

STATUSQUO

NEWINSIGHTS

Cloning andcharacterization of non-

cephalopod reflectin Generation of consensusreflectin sequence Understanding ofrelationship between primarysequence and function invitro, that can be used todevelop model for in vivofunction.

E

NDOFPHASEGOAL

QUANTITATIVEIM

PACT

Reflectin sequencedatabase is deeper thananticipated, and can provideinformation to influencefunctional studies. Reflectins can beprocessed and testedsystematically to determinestructure-functionrelationships.

Mimicry of cephalopodstructural color is impossiblewithout basic understanding ofcomponents involved. Structure-function relationshipof reflectins is unknown Limited characterization of theprotein has been performed.

Little systematic analysis ofthe protein family has beenperformed since 2004.

MAIN ACHIEVEMENTS

Identification of > 50reflectin sequences that canbe used for bioinformaticsanalysis Alignment of repeats from10 different reflectins alreadyshow high conservation incertain residues of therepeats.

Formation of thin films usingreflectin variants anddevelopment of spectralanalysis techniques

IMPACT ACHIEVED

PER(W/Y)(M/F)DMSx(W/Y)(Q/S)MDMxGR(W/Y)MDxxGR(H/Y)xxP(F/Y)

Established method for castingthin films of reflectin forcharacterization (with KaplanGroup). Determining opticalconditions for reproducibility offilms and methods to quantifyspectral characteristics of films.


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Ref1a nativereflectin Amino acid composition of Reflectin 1a

Tyr

Met

Arg

Asn

Gly

Asp

Ser

Pro

Gln

Trp

20.8

14.8

11.7

9.9

8.5

8.1

5.3

4.6

4.6

3.9

%of total

aminoacid

Phe

Glu

His

Cys

Thr

Val

Ala

Ile

Leu

Lys

3.2

1.4

1.4

1.1

0.4

0.4

0

0

0

0

%of total

aminoacid

74%

No known homologs

Reflectin Solubility Issues

Ionic detergents Ionic liquids DMSO HFIP GuHCl

Bacterial Expression of Reflectin

15% w/w 10% (w/w)

Characterize Optical PropertiesThin Films of Reflectin

Reflectins in Iridophores

reflectin 1bSubdomain 5

specular light

scattered li ht

O i l ll bili li h diff & d i 3 D i


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StatusQuo

NewInsights&Ap

proaches

Main Achievements

Quantitative

Impact

EndGoals

200 m

Leucophore ultrastructure and gross anatomyhave revealed first visualization in 3-D ofleucophores. Cell culture of leucophores hasbeen initiated. A new microscopy techniquehas been developed to simultaneouslymeasure the full spectral reflectance ofscattering thin films such as leucophores oriridophores.

Papillae gross anatomy and kinematics havebeen obtained in 3 species. Initial studies onneural control of papillae were conducted.

The ultrastructure of leucophores enablesnearly perfect diffusion of incident light.Papillae are under neurological control and theanimal is able to achieve a range of textural

states.

Research goals

Structural coloration is rarelyachieved with proteins. With AFRL.We are characterizing this yet-unidentified protein for potentialfuture applications. This study alsoaims to elucidate the biomechanicsand neural control of 3-D skinpapillae that provide dynamic textualcamouflage.

Efficient light diffusers.Commercially available diffusersrely on high-index contrast (e.g.,using titania nanoparticles), andare physically abrasiveAdaptable 3-D skin texture.3.D texture has to date not beenincorporated in moderncamouflage materials. Texture isadded in the form of additional

structures such as plantmaterials etc.

Cephalopod skin Inspiration for camouflagematerials.Leucophores appear to provideboth high reflectivity and near-Lambertian scattering with a

low-index contrast system.They are flexible and workunder wet and dry conditions.Papillae produce dynamic 3-Dskin texture along a broadcontinuum from smooth tohighly rugose. The visualsensori-motor system of rapidadaptive camouflage providesnovel bio-inspired approachesto materials science

Current impact1 Quantified spectral reflectanceand ultrastructural properties ofleucophores of one cephalopodspecies. The protein appears not tobe a known reflectin. Initial 3-Dstructure of leucophore paves wayfor accurate modeling of lightinteractions between spheres &platelets. Cell culture will enable

focused measurements2 Morphology and neural control ofindividual papillae appear complex;morphology and neuroanatomyrequire priority study at this juncture.Planned ExperimentsCharacterize, measure, & modeldifferent cephalopod & fishleucophores & papillae

Leucophore Iridophore

Optical malleability: light diffusers & dynamic 3-D texture incephalopod skin - Roger Hanlon, MBL, Woods Hole, MA

Bio-inspired approach to development of diffuse & textured camouflage materials

Leucophore whiteness:


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Purpose:

1) shadow mitigation2) maximum tonal contrast for disruptive coloration & pictorialrelief act as a light base layer upon which patterning isapplied in artificial architectures

Leucophore whiteness:(white + reflectivity)

Leucophores: passive cells that

manipulate light (i.e. no muscles ornerves to power the cells)Leucosomes: light diffusers composed ofprotein, which may be different from

iridophoresSpherical 250-1250nm dia - scattering


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LEUCOPHORES

Near-perfect diffusers of white light in all directions;

no nerves or muscles associated with them (i.e. passive)

BASIC QUESTIONS:-what combinations of leucosomes and iridophores produce different grades of

whiteness?-how do leucophores diffuse light equally in all directions?-what are the basic capabilities of leucophores from cuttlefish or octopus?


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2. PAPILLAE

How do the papillae morph in shape (biomechanics,etc.)?

What are the neural control mechanisms?How does an octopus/cuttlefish view thebackground monocularly to assess and reproduce3D texture in the skin?Can materials scientists en ineer such ca abilities?

S


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Status of achievement ofProgram Goals

LEUCOPHORES

-Aim 1: Spectrometry - New microscope and objectives acquired; isolationand culture of leucophores for high-mag spectroscopy.-Aim 2: Ultrastructural analysis - 3D structure of leucophores produced;determined temporal and spatial relationships between leucophores andiridophores.-Aim 3: Mathematical modeling - Initial model construction.-Aim 4: Protein chemistry - Leucosomes are not same reflectin protein asin iridophores; tissue samples sent to AFRL.

PAPILLAE-Aim 1: Ultrastructural analysis - Extensive skin samples obtained and

prepared (4 Octopus; 1 cuttlefish)-Aim 2: Neurophysiological control - Experiments on local and peripheralstimulation of papillae (3 species)-Aim 3: Videography - Excellent field acquisition of octopus; labkinematics from neurophysiology experiments

NEURAL REGULATION OF


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CURRENT STATUS

To understand the

neural mechanisms

& principles

underlying body

patterning in

cephalopods

NEW INSIGHTS

MAIN ACHIEVEMENTS NEXT STEPS

LONG TERM GOAL

Chromatophoreaddition aimcompleted

Good progress onFaRP role &transmitter IDaims

Process of

chromatophorematuration

NEURAL REGULATION OFCHROMATOPHORE FUNCTION IN CEPHALOPODSN. Tublitz, U. Oregon

Novel FaRP gene coding forseveral unique FMRFamideneuropeptides has beencloned and sequenced

Spatial expression pattern ofFaRP gene in cuttlefish brainhas been determined

Principles governing the

insertion of newchromatophores in the skinhave been identified

Chromatophore maturationand development have been

elucidated for all threechromatophore types

Use digitalacquisition systemto IDchromatophoretransmitters

Chromatophoremotoneuronelectrophysiology

Ai 1 N t itt d tid


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Aim 1: Neurotransmitter and neuro peptideeffects on Cuttlefish chromatophores

BEFORE AFTER


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Aim 1: Glutamate & FMRFa trigger different

responses in reddish-brown & yellow chromatophores

TMITTER CHR.EFFECT BEHAV RESP

Glutamate Fast expansion Transient

FMRFa Slow expansion Sustained

CONCLUSIONS:1) Transient patterns Glutamate

2) Sustained patterns FMRFamide

Ai 2 PTHPFRF id ff t


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Aim 2: PTHPFRFamide effectson cuttlefish chromatophores

PTHPFRFamide:1) Specificity: only black chromatophores2) Selectivity: not all black ones

3) Causes chromatophores to pulsate


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Aim 4: Regeneration study

DAY 7 DAY 12

DAY 22 DAY 30


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SUMMARY: Neural Regulation ofChromatophore Function in Cephalopods

1. All chromatophores have fast (glutamate) and slow(FaRPs) excitatory inputs

3. FaRPs have differential effects on chromatophores

2. Cuttlefish have at least 2 different FaRP genes thatencode 9 different FaRPs

4. 2nd FaRP gene expression is limited to brain regionsinvolved in body patterning

5. There are specific principles governing theinsertion of new chromatophores

6. Newly added chromatophores are the same color atfirst, then differentiate into separate color types


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SUMMARY

This represents basic research support for a broad range of

biological science initiatives to provide the background andinnovation necessary for the future development of biomaterials,devices and biosensors and their incorporation into future AirForce weapons systems.

To accomplish this, an increasing emphasis will be placed onresearch that supports the following areas: biooptics,composites, predator prey avoidance, directed self-assembly,and biotemplating.

Program Impacts:

Understanding of allosteric enzyme operation and mimicFurther understanding of Cephalopod camouflage system

Discovery of New Chromophores and their Mechanism (2010)

Importance of structural modalities in biopolymers to theiroptical and strength components (2010)

6. de long - natural materials

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