Urry D.W. (Ed.) What Sustains Life? : Consilient Mechanisms for Protein-Based Machines and Materials

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9.4 Medical Applications

525

9.4.7 Coatings for Medical Devices to

Prevent Adverse Reactions and

Improve Function

Before proceeding to the advanced technology

area of biosensors, brief consideration goes to

coatings of medical devices to improve perfor-

mance and to prevent such adverse reactions as

adhesion of the medical device to the tissues,

bacterial adhesion, platelet adhesion and acti-

vation, and adverse tissue reaction of host to

device from the standpoint of protecting both

the host and the function of the device.

9.4.7.1 The Cardiovascular Example of

the Utility of Coatings

Coatings of devices involved in cardiovascular

intervention include (1) the vascular prosthesis

itself,

(2) cardiac catheters, (3) cardiac (coro-

nary artery) stents, (4) leads for pacemakers, (5)

tubing for cardiac assist devices, (6) temporary

artificial pericardium following open heart

surgery, (7) tubing for continuous ambulatory

peritoneal dialysis, and (8) drainage tubes. Of

course, the coatings may simultaneously func-

tion as delivery vehicles for the whole range of

therapeutic agents.

9.4.7.2 Coatings to Improve Urinary

Indwelling Catheters

Perhaps the medical device that has had longest

term need for coatings to improve function is

the urinary catheter and more specifically the

urinary indwelling catheter. Approximately

one-fourth of the patients in chronic care and

geriatric facilities utlilize urinary indwelling

catheters. Shorter term use follows prostatic

surgery, considered the most frequent surgical

procedure in men over 65 years of age, and

attends cardiovascular and other surgical pro-

cedures. It has been indicated that 15% of hos-

pital admissions require indwelling urinary

catheters for one reason or another.

9.4.7.3 Consequences of Indwelling

Urinary Catheter Use

The problems arising out of urinary indwelling

catheter use are niany: (1) They are the source

of as many as 30% of all hospital acquired

infections. (2) Urethral strictures result from

abrasions due to insertion, shifting, and

removal, from bacterial colonization of the

abrasions, and from inflammatory reaction to

toxic substances that leach out of the catheter.

(3) Long-term usage has been credited with

causing urethehal hyperplasia and dysplasia

and bladder tumors. (4) Problems of luminal

encrustation that block flow and catheter

replacement aggravate the foregoing list of

adverse consequences.

Obviously, the adverse results of increased

morbidity, mortality, duration of hospitaliza-

tion, and overall health care costs argue for the

small investment in coating technology to

correct this situation. Just as clearly, elastic and

plastic protein-based polymers could ehminate

the majority of these adverse consequences.

9.4.8 Biosensors (e.g., Diagnostics)

A particularly exciting new application is under

way. It is the development of nanosensors with

the ultimate potential for sensitivity, that is, the

detection of single molecule interactions. It is

expected that a single molecule of phosphate,

nerve gas, toxin, and explosive, for example,

TNT and the associated DNT, could be

detected. The most direct approach to demon-

strate this sensitivity and at the same time to do

so with high selectivity is to detect a single

phosphorylation event, as discussed below. First

however, a short description of the atomic force

microscope that, when suitably modified and

used with designed elastic protein-based poly-

mers,

enables a high level of detection.

9.4.8.1 Atomic Force Microscopy in the

Single-chain Force-extension Mode

9.4.8.1.1 The Atomic Force Microscope

The atomic force microscope (AFM), as devel-

oped by Paul Hansma, constitutes a cantilever

with a very fine tip that scans over a surface. By

measuring changes in the force between tip and

surface, the cantilever tip, as it scans or rasters

over a surface, forms an image of the surface and

of molecules, for example, polymers, on the

surface. The image arises out of the capacity to

detect extremely small changes in the force of

526 9. Advanced Materials for the Future

photo diode

micrometer

screw

laser diode

piezo with

strain gauge

microscope objective

on CCD camera

FIGURE 9.51. AFM spectroscopy designed for a single-chain force-extension mode; diagram of the instru-

mental set-up of Gaub and coworkers.^^^'^^ (Reproduced with permission from Urry et al.^^)

interaction of tip with surface and to do so with

remarkable spatial resolution. What can happen

to complicate the imaging of macromolecules on

the surface is that these polymers can stick both

to the tip and to the surface, and then the force

of extending the molecule along the surface

smears the molecular image being sought.

This compUcation, however, becomes the

basis for a new and equally interesting mea-

surement of special interest to elastic protein-

based polymers. For our particular interest the

usual AFM instrument is modified, as advanced

by Hermann Gaub and coworkers,^^^'^^ so that

the cantilever tip moves in the direction per-

pendicular to the surface (Figure 9.51 presents

a diagram of the instrument). With polymer

attached at one end to the surface and at the

other to the tip, the force as a function of exten-

sion of the molecule is obtained.

9.4.8.1.2 Two Distinct Profiles of a

Single-chain Force-extension Curve

Of direct interest to us are two distinct profiles

of the force versus extension curve. The first

is obtained for our bioelastic polymers that

exhibit near-ideal elasticity,^^'^^ which is a

simple reversible monotonic curve of ever

increasing force with extension until detach-

ment occurs with the drop of the force to zero.

The second is the characteristic sawtooth

profile for titin, the energy absorbing elastic

filament of muscle, which is a protein-based

polymer of about 100 similar globular protein

units of about 100 residues.

The extraordinary work of Gaub and Fer-

nandez on titin, as represented by short strings

of composite globular elements, is shown in

Figure 9.52.^^^ Figure 9.52A shows four globu-

lar elements and depicts the unfolding of a

single globular element due to extension, and

Figure 9.52B shows the experimental sawtooth

pattern resulting from the sequential unfolding

of a series of six globular elements followed

by the detachment of the chain from either

substrate or cantilever tip with a return to zero

force. Thus, the unfolding event for each glob-

ular element is seen as a single tooth, and each

tooth can be fitted with the mathematical

expression of the worm-like chain model. The

differences between extension limits for each

tooth, as measured by fitting to the equation for

a worm-like chain, provides a measure of the

increase in extension length due to the unfold-

ing of a single globular element. This difference

provides a measure of the number of residues

comprising the globular element that was

unfolded (see Figure 9.52B).

9.4 Medical Applications

527

AFM

Cantilaver

Recombinant

TItIn Fragment

50 100 150 200 250

Extension (nm)

FIGURE

9.52. Atomic force microscopy, single-chain

force-extension experiment. (A) Drawing of the

extension of four globular subunits of a titin mole-

cule shown as the stepwise unfolding of the string of

globular domains. (B) Sawtooth pattern in the force-

extension curve due to serial unfolding of titin glob-

ular domains, fitted to a worm-like chain model that

indicates an approximate

nm extension as each

domain unfolds. (Reproduced with permission from

H.E. Gaub and J.M. Fernandez.^°^)

528

9. Advanced Materials for the Future

9.4.8.2 Nanosensors to Detect

Kinase Activities

9.4.8.2.1 One Particular Design of

a Nanosensor

One nanosensor construct of interest to us com-

bines the two elastic materials by positioning

the sequence for a single approximately 100

residue globular protein element between two

sequences of near-ideal elastic protein-based

polymer. The particular globular element uti-

lizes the fibronectin type III domain of tenascin

that contains the GRGDSP cell attachment

sequence at the turn connecting the F and G P-

strands, as represented in stereo in Figure 9.53 A

in the ribbon representation and in Figure 9.53B

Phosphorylation site 4

FIGURE 9.53. Stereo views of a fibronectin type III

domain of tenascin with aromatics (black), other

hydrophobics (gray), neutrals (light gray), and

charged residues (white). (A) Ribbon representation

showing seven p-strands and the turn containing the

GRGDSP cell attachment site that in our designed

sensor is replaced by RGYSLG to provide a phos-

phorylation site as approximately located. (B) Vine

representation showing side chains. (Prepared using

the crystallographic results of Bisig et

al.^°^

obtained from the Protein Data Bank, Structure File

ITEN.)

9.4 Medical Applications

529

in the vine representation that shows the side

chains.^^^ The globular fibronectin type III

domain is a seven-stranded, hydrophobically

folded, P-barrel or sandwich that is one of the

two types of globular domains present in titin

and that also occurs in tenascin for which a

crystal structure is available (see Figure 9.53).

In Figures 9.35 and 9.37 the GRGDSP cell

attachment sequence was used within elastic

protein-based polymers, such as [(GVGVP)io

GVGVPGRGDSP(GVGVP)io]i8(GVGVP),

to induce tissue generation. The GRGDSP

sequence is found in the 99 residue globular

protein of Figure 9.53, known as the fibronectin

Type III domain (Fn3). For the sensor apphca-

tion the sequence, RGYSLG, replaces the

GRGDSP, to give the globular protein desig-

nated as Fn3' (GRGDSP^RGYSLG), which is

placed in an elastic protein sequence to give

[(GVGIP)io]io-Fn3'(GRGDSP^RGYSLG)-

[(GVGIP)io]n2- A cartoon of this construct,

truncated in terms of the length of the repeat-

ing pentamer sequences, is given in Figure 9-54.

This single molecule construct would be

expected to give a force-extension curve with a

single sawtooth due to the unfolding of a single

globular element superimposed on the simple

monotonic curve of the nearly ideal elastic ele-

ments, as shown in Figure 9.55A. The capacity

to sense, in this case the single molecular event

of phosphorylation of the serine side chain,

requires that the single sawtooth change its pro-

file in a reproducible way on interaction of the

globular sensing element with the "analyte."

9.4.8.2.2 Assay of Kinase Activities for

Diagnostics in Tumor Tissues

As the starting point for this development, we

chose to design for detection the very polar

phosphate group of biology that is capable of

exerting one of the largest apolar-polar repul-

sive forces of interaction. The site for phospho-

rylation is the serine (Ser, S) residue in the

peptide sequence RGYSLG, a phosphoryla-

tion site for the cardiac cyclic AMP-dependent

protein kinase. This sequence replaces the

GRGDSP cell attachment site within a 99

residue globular protein element of the tenth

type III domain of fibronectin. The particular

GRGDSP-^RGYSLG

FIGURE

9.54. Cartoon of a molecular construct

(in stereo) representing [(GVGIP)io]ni-Fn3'

(GRGDSP->RGYSLG)-[(GVGIP)io]n2 strung

between a cantilever tip and the surface of an

atomic force microscope designed for single-chain

force-extension studies. The molecular construct is

designed with a globular sensing element sand-

wiched between stretches of elastic protein-based

polymer in the p-spiral structure. The globular

sensing element is the 99 residue tenth fibronectin

type III domain (Fn3') in which the cell attachment

sequence (GRGDSP) has been replaced by a site for

phosphorylation (RGYSLG) at the location indi-

cated by the oval. (The globular element utilizes the

crystal structure of

Bisig

al.^^^

as obtained from the

Protein Data Bank, Structure File ITEN. Courtesy

of Bioelastics Research, Ltd.)

530

9. Advanced Materials for the Future

gene construct utilized for preliminary exami-

nation resulted in expression of the sequence

[(GVGIP)io]ni-Fn3'(GRGDSP->R-

GYSLG)-[(GVGIP)io]n2 with nl = 16 and n2 =

6, that is, 160 pentamers on one side and 60 on

the other side of the globular sensing element.

^ 400-

^300.

-$"200-1 unphosphorylated

100-

150 200 250 300 nm

AFM force-extension (fAL) mode

unphosphorylated

phosphorylated

1 X 103 frequency

AFM DFS (isometric)

FIGURE 9.55. Proposed means whereby elastic

protein-based polymers can be used with special

adaptations of the atomic force microscope to detect

single molecular interactions. (A) Single-molecule

force-extension curve obtained for [(GVGIP)io]ni-

Fn3'(GRGDSP-^RGYSLG)-[(GVGIP)io]n2, where

the 99 residue globular sensing element

Fn3'(GRGDSP-^RGYSLG) is the tenth fibronectin

type III domain in which the cell attachment

sequence (GRGDSP) has been replaced by a site

(RGYSLG) for phosphorylation by cardiac cyclic

AMP-dependent protein kinase; also with nl = 16

and n2

Fit to a worm-like chain model consistent

with the unfolding of the Fn3' globular domain. The

dashed curve is the anticipated result should phos-

phorylation completely hydrophobically unfold the

globular sensing element as suggested by the data in

Figure 9.56. (Unpublished results of T. Hugel, M.

Seitz,

Gaub,

Pattanaik, and

D.W.

Urry) (B) Pro-

posed dynamic force spectroscopy curves of single

twisted filament containing a site for enzymatic

phosphorylation (illustrated in Figure 9.54) before

(above) and after (below) phosphorylation that

assumes the acoustic absorption curves in Appendix

A, Figure 7A.

The single-chain force-extension curve

for [(GVGIP)io]i6-Fn3'(GRGDSP

-^RGYSLG)-[(GVGIP)io]6 given in Figure

9.55A derives from collaboration withT. Hugel,

M. Seitz, and H. Gaub. We tentatively interpret

this curve to be the stretching out of the bioe-

lastic sequences until the force is sufficient,

about 250 pN, to unfold the single globular

domain, as shown in Figure 9.52B. The increase

in force then resumes until it is sufficient to

cause detachment from either cantilever tip or

surface. At this point, about

300

nm extension,

the force drops to zero.

By fitting the curve with the mathematical

expression for a hypothetical worm-like chain,

an estimate of the change in length of the glob-

ular element is possible. In the case in Figure

9.55A, the change in length is as expected

for the unfolding of a 99 residue globular

protein.^^^ Thus, from the criteria of both the

force at which unfolding occurs and the length

change that occurs on unfolding, it is not unrea-

sonable to consider this the sawtooth due to

unfolding of Fn3'(GRGDSP^RGYSLG). We

expect that phosphorylation of this single

RGYSLG site in the globular domain will

change the force-extension profile for unfold-

ing. If this does occur, then we will have

detected a single molecular event, the phos-

phorylation of a single site in a single globular

protein domain.

9.4.8.2.3 Effect of Phosphorylation of

[(GVGIP)io]i6-Fn3'(GRGDSP->RGYSLG)-

[(GVGIP)io]6 on Hydrophobic Association

The temperature profile for turbidity forma-

tion of [(GVGIP)io]i6-Fn3'(GRGDSP->R-

GYSLG)-[(GVGIP)io]6 in Figure 9.56A shows

a simple profile for aggregation by hydropho-

bic association. On phosphorylation by cardiac

cyclic AMP-dependent protein kinase, how-

ever, the profile becomes more complex with a

delayed aggregation occurring at a 20°C higher

temperature (see Figure 9.56B). Furthermore,

two-dimensional nuclear magnetic resonance

(2D-NMR) studies show that phosphorylation

disrupts hydrophobic association within the

globular sensing element (L.C. Hayes and D.W.

Urry, unpublished data). In particular, the

9.4 Medical Applications

531

[(GVGIP)io]„i-Fn3'(GRGDSP^RGYSLG)-[(GVGIP)io]n2

16andn2 = 6

125-

lOOH

TCO

125-

100 H

Tj=15.4°C(0hrs)

Tj=15.4°C (4hrs)

Tt=15.6°C(24hrs)

Tj=15.9°C(72hrs)

Tt=35.5°C (4 hrs)

Tt=34.7°C (24 hrs)

Tj=35.8°C (72 hrs)

20 30

T(°C)

FIGURE 9.56. Determination of Tt before and after

phosphorylation of the designed protein-based

polymer sensor [(GVGIP)io]ni-Fn3'(GRGDSP^

RGYSLG)-[(GVGIP)io]n2, with nl = 16 and n2 = 6.

The presence of

phosphate in 1,199 residues at the

RGYSLG site of the 99 residue globular protein

markedly increases Tt of either about 1/2 of the

polymers, that

is,

K ~

or causes initial formation of

micelles followed by association of the micelles.

(UnpubHshed

results,

Pattanaik,

T.M.

Parker, and

D.W. Urry.)

NOESY (nuclear Overhauser enhancement

spectroscopy) plot for the aromatic region

before phosphorylation of (GVGIP)i6o-Fn3'

(GRGDSP-^RGYSLG)-(GVGIP)6o contains

many cross-peaks, many proton-proton con-

tacts involving aromatic residues. These cross-

peaks report the hydrophobically folded p-

barrel, as represented in Figure 9.53 for the

structure of the tenascin fibronectin type III

domain, as there are no aromatic residues in

532

9. Advanced Materials for the Future

(GVGIP)n. Phosphorylation removes more

than two-thirds of the proton-proton contacts

that report the hydrophobic associations within

the p-barrel.

Accordingly, both the temperature profile

data for following aggregation by hydrophobic

association and the 2D-NMR data indicate that

phosphorylation disrupts hydrophobic associa-

tion within the globular sensing element. Phos-

phorylation at one end of the p-barrel, as shown

in Figure 9.53A, reaches through the P-barrel

and, at least in part, disassembles the hydropho-

bically assembled structure. Thus, phosphoryla-

tion disrupts hydrophobic association, as a clear

demonstration of

AGap,

the apolar-polar repul-

sive free energy of hydration. The result in the

single-chain force-extension curve would be a

change in the profile of the single sawtooth.

Construction of a gene and expression of the

protein-based polymer for stable attachment to

cantilever tip and substrate, which means intro-

duction of proper functional groups at each end

of the designed polymer, will allow for a rigor-

ous testing of this nanosensor with potential to

detect a single molecular interaction.

9.4.8.2.4 Value of Sensitive and Selective

Assays for Kinases and Phosphatases

As is apparent in Chapter 8, phosphates are

the most polar molecular species available in

biology for controlling hydrophobic association/

dissociation, that is, for controlling processes

that occur by inverse temperature transition. It

follows, therefore, that kinases for phosphoryla-

tion and phosphatases for dephosphorylation

would be fundamental to key cellular trans-

ductional and transformational processes. ^^^'^^^

Often in cancerous and other diseased states,

the activities of these enzymes are abnormal.

Importantly for our interests, their sites of inter-

action can be very selective. Changes in protein

kinase C activities have been reported to be

abnormal in colon, breast, and skin cancers.

Protein tyrosine kinase, which selectively phos-

phorylates the internal tyrosine (Tyr,Y) residue

in the sequence GIYWHHY, is overexpressed

in colon and breast cancer.^^^^^^ Further-

more, cycUc AMP-dependent protein kinase

activities have been associated with the onset of

Alzheimer's disease, where the proteolytic

degradation product of a hyperphosphorylated

former membrane protein is the principal con-

stituent of the characteristic plaques of

Alzheimer's disease.^^^ Thus, there is great

interest in the capacity to assay for kinase activ-

ities in tissue homogenates,^^"^^^^ particularly if

simple biopsies could suffice and there would

be no need to resort to phosphorus-32, a high-

energy emissions radionuclide that increases

health risks.

The potential, therefore, is to introduce single

or multiple globular protein element(s) with all

of the specificity of an enzyme and to achieve the

ultimate in sensitivity, the detection of a single

molecular interaction. By selection of the appro-

priate globular sensing element, one can look to

the polar analytes of phosphates, nerve gases,

toxins, or explosives that

would,

on interaction

with the globular protein sensing element, alter

its sawtooth profile.

9.4.8.3 Nanosensors Using

Mechanical Resonances

Another striking property of certain elastic

protein-based polymers, as shown in part by the

data in Figure 9.15, is that they can exhibit an

intense mechanical resonance in the acoustic

absorption range that varies as a function of

hydrophobic assembly. That is, the intensity of

the mechanical resonance increases as the

hydrophobic assembly of the inverse tempera-

ture transition progresses and, of course,

decreases as hydrophobic dissociation occurs.

Thus,

elastic protein-based (bioelastic) poly-

mers are capable of 18 classes of pairwise free

energy transductions; they can be produced

with diverse composition, fixed sequence, and

precise length, and they can exhibit intense

mechanical resonances in the acoustic absorp-

tion range that undergo very large intensity

changes during transduction.

Interesting additional methodologies are

under development; they build on the funda-

mental design of the AFM. This development

involves vibrating the cantilever at a frequency

kHz, for example, to characterize the

shear modulus properties of the polymer with

which the cantilever tip makes contact, as first

9.4 Medical Applications

533

considered by Mervyn Miles and colleagues in

Bristol. With the typical rheological instrumen-

tation, as in obtaining the loss shear modulus

data in Figure 9.15, it is not possible to reach

frequencies greater than several hundred

Hertz. On the other hand, both the acoustic

absorption data of Lev Sheiba and the low-

frequency dielectric relaxation data (loss

permittivity data) in Figure 9.15 argue that a

mechanical resonance occurs centered near 3

kHz, and this is supported by the overlap of the

loss shear modulus and loss permittivity data in

Figure 9.15. With proper instrumental design, it

is considered possible to assess the loss shear

modulus at frequencies below the exciting fre-

quency, for example, below

kHz.

Such measurements, first suggested to us by

Vlado Hlady of the University of Utah, provide

an opportunity to reaffirm the mechanical res-

onance in the acoustic frequency range near

3 kHz. Importantly, such an instrumental capac-

ity coupled with elastic protein-based polymers

that exhibit acoustic absorption would consti-

tute nanosensor development using isometric

conditions, that is, at fixed extension. With the

appropriate sensing filament strung between

cantilever tip and substrate, any transductional

interaction with the filament, that is, that

changed the extent of hydrophobic association

within the filament, would be detected as a

change in the intensity of the absorption, as

represented in Figure 9.55B.

Without the need to stretch the elastic

sensing element, a robust device could be con-

structed that would be deployable for use in the

field. One design of the sensing element for

such a device is shown in Figure 9.57. Vlado

Hlady has built a measuring device capable of

what he calls dynamic force spectroscopy, and

we look forward to future opportunities to

develop a range of useful nanosensors capable

of the ultimate in sensitivity that would be

useful in medical diagnostics and in detection

of terrorist threats.

9.4.9 Biodegradable Plastics

Certain compositions of plastic protein-based

polymers can be prepared that melt rather than

decompose, as protein materials normally do

on excessive heating. In the melted state they

can be pulled into fibers. One such composition,

poly(FE(Boc)GVP), is shown in Figure I.IOA

and compared with similarly formed fibers of

more commonly known plastics, polypropylene

(Figure 1.1 OB) and polystyrene (Figure I.IOC).

One advantage of the bioplastic polymers is

that they can be programmed for different

rates of proteolytic degradation with half-lives

ranging from days to decades. These properties

make them of interest for a number of applica-

tions,

for example, as sutures and staples to be

used in closing following surgical procedures

and even as bone screws for orthopedic appli-

cations. Additionally, such biodegradable

plastics could be used as both structural and

controlled release devices, as in the delivery

of bone morphogenic proteins for bone

reconstruction.

9.4.10 Sound Absorption for

Hearing Protection

Certain bioelastic compositions, when in the

folded and assembled state above the temper-

ature of the inverse temperature transition, to

the best of our knowledge provide materials

with the highest sound absorption per unit

volume. One such composition is compared in

Figure 7A of Appendix A to standard materi-

als (natural rubber and polyurethane) used for

sound absorption. The lower flat curve repre-

sents the absorption by natural rubber over the

frequency range of sound, the acoustic absorp-

tion range. As we understand it, the somewhat

better absorbing, low, flat curve is due to a very

favorable sound absorbing elastic material

made from petroleum. The mountainous-

looking curves are due to a bioelastic material.

Below the transition temperature of the

polymer, the bioelastic polymer is definitely

better, but only by a factor of two or so. On

raising the temperature above the onset of the

transition temperature, the sound absorption

increases dramatically and would probably

double on increasing to body temperature.

Among other things, the property of these

materials appears to be favorable for hearing

protection, for example, when made into an

earplug or ear

muff.

534

9. Advanced Materials for the Future

Attachment, below T , of

amine from each of

three chains to the activated surface somewhere

within a 20 nm hydrodynamic diameter of

300 kDa bioelastic construct

Attachment of Au-coated cantilever

tip to SH of bioelastic construct

Lower

below operating

temperature

(e.g.

by addition

salt)

during cyclic extensions

to form twisted filament

Elastically extensible

to several hundred nm

FIGURE

9.57. (A) Representation of amorphous

triple-stranded protein-based polymer below Tt. (B)

Cross-linked SH groups at one end of the three

chains of the amorphous triple-stranded protein-

based polymer attached to a cantilever tip and the

amino functions at the other ends of the three chains

attached to the substrate. (C) The expectation is that

twisted filament

width

r""

)

; ^

( \

J \

?2V

^ ^

pil

^^i^v^

-"]

-^y

Surface activated

for amine attachment

a series of extension/relaxation cycles will draw the

three chains into a twisted filament. This twisted

filament structure is expected to exhibit an acoustic

absorption spectrum in a similar frequency range as

that in Figure 9.58 and to change on phosphorylation

as indicated in Figure 9.55B.

References

L. Pasteur, "Pourquoi la France n'a pas trouve

d'homme superieurs au moment du peril." Rev.

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J. Bronowski, The Ascent of

Man.

Little, Brown

and Company, Boston, 1973, p. 110.

D.W. Urry, R.D. Harris, and K.U. Prasad,

"Chemical Potential Driven Contraction and

Relaxation by Ionic Strength Modulation of an

Inverse Temperature Transition." /. Am. Chem.

Soc, 110, 3303-3305,1988.

Praemer, S. Furner, and D.P. Rice, Muscu-

loskeletal Conditions in the United States. Park

Ridge, American Academy of Orthopaedic

Surgeons, 1992.

G. Waddell, "Low Back Pain: A Twentieth

Century Health Care Enigma." Spine, 21,

2820-2825,1996.

6. T.W. Hu, "Impact of Urinary Incontinence on

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Report/Abstract—National Pressure Ulcer

Advisory Panel, Annual Conference Program

2001.

NPUAP 11250 Roger Bacon Dr., Suite 8,

Reston,VA 20190-5202.

8. National Pressure Ulcer Advisory Panel, State-

ment on Pressure Ulcer Formation and Pres-