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					Bridging nonliving and living matter:

----- Experimental   approaches towards the creation
of a protocell


                     Liaohai Chen

                  Argonne National Laboratory

                 Rush University Medical Center
big picture

What is life?
Where do we come from?
                                  understanding modern
Are we alone?                             cells



               assembly of life
              in the laboratory



                                          search for
                                       life elsewhere
How can minimal life be useful?
                                                  our vision…
Overall experimental approaches:
---- top down vs. bottom up

                 modifying modern
                       cells

Pathways
 to novel                               hybrid
                    assembly of
   living                                living
                Bio/organic materials
 matters                                matters


                 inorganic replicator
                      systems
Top down approach (C. Venter and H. Smith ):
                   Stripping down a modern cell

   Mycoplasma genitalium:
   Living Organism
   With the Smallest Genome




                                Minimal life format
                                with only 265 genes from
                                original 486 genes
Bottom up: create a living cell

Experimental Approaches:

Cell mimic:
Using modern cell apparatus (such as enzymes)
       RNA world, Artificial Cells ……


Cell inspired:
Using relative simple organic molecules (protocell)
        PNA, Peptide, and PAH Worlds
Synthesizing life in RNA world                 Szostak et al. 2001




                                Vesicle growth and division:
                                Self-relicating membrane vesicles can grow
                                either gradually or in discrete steps, and
                                may divide either spontaneously or under
  Vesicle growth and division   the influence of external forces
RNA based artificial cell
 RNA World Hypothesis
      - Catalytic activity (= Ribozymes)
      - Ligand-binding activity (= Aptamers)                 like proteins
      - Regulation activity (= Riboswitches)
      - Depository of genetic information                    like DNA today


                            DIRECTED            Catalysts
              Catalysts   POLYMERIZATION
Substrates
               Genes
                            MEMBRANE
                             GROWTH              Genes
   Energy


                                                                     Catalysts
                                    Catalysts
                                                 Catalysts            Genes
                                     Genes
                                                  Genes


 No metabolism - all substrates are provided. (Scavenging mode)
 No energy requirements - energy is present in
 chemical bonds of substrates.
A vesicle bioreactor as a step toward
an artificial cell assembly




                         Vincent Noireaux and Albert Libchaber,
                         Rockefeller University,
                         PNAS, 2004, 101, 17669-17674.
Bottom up: create a living cell from scratch
Using relative simple organic molecules to
create a protocell
 (1) European Commission grant on Programmable Artificial Cell Evolution (PACE)
 14 institutional partners including Argonne.
 http://www.protocell.org/PACE
 Task: Explore the utilization of the simplest feasible elementary living units to build
 evolvable nano-systems based on microfluidics


 (2) Los Alamos LDRD-DR grant on Protocell Assembly (PAs)
 http://protocells.lanl.gov
 Task: Explore the underpinning since for the assemble of a minimal self-replicating
 nano-machine


 (3) WPAFB USAF grant on simulation of Cell-Like Entities (CLE)
 Task: Indentify best mesoscale simulation techniques for CLE investigations.
               • European Commission grant
PACE =           (E8.5M, 4 years) on chemical
Programmable     program-mability (IT), with 14
Artificial       institutional partners (universities,
                 government labs, and city
Cell             government).
Evolution      • Task: Explore the utilization of the
                 simplest feasible elementary living
                 units to build evolvable nano-
                 systems based on microfluidics.




                                         protocells - big picture…
                                            Evolutionary design
                                            and programming of
                                            protocells using
                                            programmable microfluidic
                                            silicon chip technology.

                                                              The W -machine components


                                                                          Electronic                    Software
Reconfigurable BioModule with a complex                                   Computer                      Interface
 programmable logic device (CPLD) and
        integrated microfluidics.

                                               Reconfigurable                                    Monitoring
                                               Electronic                                        System
                                               Interface



                                                               Parallel                  Microfluidic               Chemical
                                                               Actuator                  Network                    reservoirs
                                                               Network


                                                                                                 Product



                                          FET Complex Systems, 14.1.03                 key protocell components…
Los Alamos National Laboratory

Protocell Assembly Program (PA)             living matter
                                          modified natural cells
PA program: science &
mission vision                            artificial cells

                                          programmable protocells
to assemble a minimal living system
(protocell) from inorganic and            protocells
organic components
                                          self-reproducing aggregates

to utilize protocells as the foundation   metabolic units
for a living technology based on
                                          templating units & polymers
robustness, autonomy, local
intelligence, self-repair, adaptation,    self-repairing crystals
and self-replication.
                                           nonliving matter
                                                             what is life…
PA Proto-cell
                                 container
                                 (localization)




           metabolism                                         genes
           (energy transformation)                          (inheritance)

Systemic design principle:

1) Minimize the structures for the required cooperative functionalities;

2) Organizational closure: cooperation of aggregates;

3) Thermodynamic and kinetic complementary

                                          Rasmussen, Chen, Nilsson & Abe, Artificial Life 7 (2003) 269
Multiscale simulations
PA Proto-cell

                  proto-container




  proto-metabolism                  proto-genes

• Organizational closure: cooperation of aggregates
• Thermodynamic and kinetic complementary
Candidate for proto-container

        Phosphlipids            Fatty acids bi-layer vesicles




                                Vesicle from nonanoic acid
                                  David Deamer, UCSC, 2002
                                 Fatty Acid Assemblies
Experimental Details
•   0.06g OAcid/MeOH Stock(1655mmol OAcid)
+0.24g Nile Red in MeOH, (164mmol NR red)
MeOH evaporated
+2.5g Bicine/Na Buffer (0.2M Bicine, pH~8.5)

•      5ml of the above solution imaged with a 100x oil immersion
       objective on an inverted fluorescence microscope
                                                                    Oleic Acid, Cis-9-octadecenoic acid

        25 mm
                                                                              5 mm




    Sample ID:PC7


                                                                         Doshi & Shreve (2005)
Fatty (oleic) acid vesicles
 (micelles for higher pH)
   Anchoring Vesicle on the surface


                                    streptavidin         streptavidin
                                       (FITC)               (FITC)

                                        biotin               biotin




                                   passivated, partially biotinylated,
                                   patterned, self-assembled monolayer

Can now attach biotinylated species to patterned film
                                         Oleic Acid vesicles (pH 8.5)
                                         with 0.2 mole% biotin-PE
~30 mm                                   lipid




                                          Boundary of
                                          streptavidin
                                          pattern




         Loading hydrophobic molecules
         ------ Nile Red
Achieve simple self-replication
      OCH3
                 O
                                 h
             O



       NO2
                      OCH3
                             H
                                           O
                             O
                                 +    HO


                       NO2
PA Proto-cell

                  proto-container




  proto-metabolism                  proto-genes

• Organizational closure: cooperation of aggregates
• Thermodynamic and kinetic complementary
                                    Design principle
    A               B                                                    A B
                            B                                           B     B
                B
                                                                        A
                             B                                          BA      B
        A       B
                    B B                                                     B A
            A                   A
                         A




                                                              B                     B
    B                            B                                  B
            B                         B                  B                      B         B
B                       B
                                                         B          B                     B
B           B           B             B                                         B
                                                              B B                   B B
    B B                         B B
                                           Light / Chemical
                                A                                       B
                        Precursor         “Gene” / catalyst (P)         Lipid
A              B       nB


                          PACE
          PA



nB    +            P    2P


       +           P      P   +   P’
nB



     P: PNA and Peptide
Rational design of “gene controlled metabolism”

Lipid molecules can be generated by photo induced electron
transfer fragmentation reaction


           The yield is controlled by charge separation



If certain PNA or peptide sequence can function as electron relay


   Then “genetic” material will control the lipid formation


           Thus control the container replication.
Lipid molecules can be generated by fragmentation reaction
                                  _
        O                                                           O
                   O                         BDEanion radical                               _       O
        C    CH2 O C (CH2)11      CH3                               C     CH2       +       O C (CH2)11         CH3
                                            (Negative value)



                   ~ 5 kcal/mol
                 Reduction Potential                                               ~ -80 kcal/mol
                                                                                - Electron Affinity


         O          O                                           O
                                             BDEneutral                                         O
         C   CH2 O C (CH2)11      CH3                           C       CH2     +       O C (CH2)11           CH3
                                            ~ 70 kcal/mol




                        O               O                                           _   O
                        C   CH2       O C (CH2)11     CH3                           O C (CH2)11         CH3




                   Hydrophobic                                                      Amphiphilic
                   phenacyl ester                                                   carboxylic acid
 Anion radicals can be generated by photo-induced electron transfer


                                                                  _
                kq           . _
                             +  .           kce           .
                                                          +       .
D* +       A                D , A                        D    +   A




               k-et                                 kr
                                 krcp




 D     +   A                             Products
Phorto-fragmentation reaction of phenacyl ester via photo-induced electron transfer
                      O          O                                         O                 O
    D*        +       C    CH2 O C (CH2)11    CH3              D           C     CH2 O C (CH2)11               CH3




        Vis

                       O          O                                                          O
                                                                                                                 O
     D        +        C   CH2 O C (CH2)11    CH3                  D       +                 C     CH2         O C (CH2)11   CH3




                                                                                         O
                                                                                                                     O
                                                                                         C       CH3       +     HO C (CH2)11      CH3




  Overal reaction:


                  O          O                                         O
                                                    light                                              O
                  C   CH2 O C (CH2)11   CH3                            C       CH3   +       HO C (CH2)11            CH3
                                                    Electron
                                                    Donor
Photo-driven production of lipids
                                 O                                                                         O
                                                 O                                                                           O
           *
           D   +                                                                          D
                                 C    CH2 O C (CH2)11

                                             A
                                                              CH3

                                                                 98 %
                                                                                                           C   CH2 O C (CH2)11           CH3
                                                                                                                                                         (a)
                                                                                                               2%
                                  O                                                                                O               O
                                                 O
           D       +              C    CH2 O C (CH2)11        CH3                             D   +                C    CH2      O C (CH2)11       CH3



                                        OH OH
     D =       N                                                       N
                                        C   C                                                                      O                      O
                                                                                      N
                                      H3C   CH3                                                                C       CH3            HO C (CH2)11          CH3


     (b)
           60000                                                                                      When the sensitizer (CCl4) is presented:
                               Fluorescence of ketone product
      Flu
           50000
                                                                                                       *
                                                                                                       D       +       A                  D        A
      ore
      sc 40000
      en                                                                                                                         5%                     CCl4
      ce                                                   Quantum yield is low!!                                                                 95%
      Int 30000                                                                                              A
      en                                                                                               D       +
      sity
           20000                                                                                                                              D    +    A    + CCl4


           10000
                       Formation of ketone via
                       photo-induced fragmentation reaction                                                        (c)                 Ketone product
               0
                         500           550           600            650               700

                                             Wavelength (nm)


                                                                                      Rasmussen, Chen, Nilsson & Abe, Artificial Life, 9 (2003) 269
Key components for effective lipid production via photo-
fragmentation reaction:

                Electron Donor (Sensitizer)

                Electron relay system


           PhCOCH2-OCOC11H23    D
                                         Electronic Relay   Electron
                                                            Barn
          PhCOCH2-OCOC11H23     D



                         COOH       h
Selection of sensitizer
    1.   Its excited state are capable of reducing phenacyl ester;
    2.   Its cation radical is capable of oxidizing adenine or guanine;
    3.   Synthetically feasible to be linked to PNA backbone;
    4.   Robust


Potential organic molecules as sensitizers

                  COO
                                                                     CH2CONH
                         Ru (PF6)2         N
          N   N      3


 Excited state reduction potential: -0.81 V; capable of reducing phenacyl ester

 Oxidation potential for Ru(bpy)33+/2+ : 1.29 V
 capable of oxidizing adenine (1.1 V) or guanine (0.8 V)
Certain sequence of XNA or peptide can function as an electron relay
system.
  Electron transfer via DNA --- well demonstrated in the literature
  Example I:
  Charge can be generated by quenching the fluorescence of sensitizer directly by DNA
                                         *
                                             CONH    G    DNA
          HNOC



                                                                                       CONH   G   DNA
                                                       HNOC



                              Lewis, et al. JBIC, 1998, 3(2), 215
 Example II:
 Charge can be generated by relaying electrons from cation radical

                                                 kq = 3.3 x 09 M-1S-1
                                     +       A                                                +    A
                                O                                       O          O   O
            O             O
                   OCH3                                                     OCH3



                Johnston et al, photochem. & photobio. 2000, 72(2), 155
     DNA (PNA) charge transfer efficiency
                                       (after Alexander L. Burin, 2003)




    Kalosakas, Rasmussen and Bishop,
     J. Chem. Phys., 118 (2002) 3731




Expected charge transfer rates
     in PNA around 1011 s-1
 (less water and ions in lipids)
                                       Integrated protocell metabolism …
 Chemistry for surfactant production -- I


                     O           O                                O               O
       *
       D   +         C     CH2 O C (CH2)11   CH3    D             C       CH2 O C (CH2)11       CH3



                                                                              H
                                             65%                  H2N         N   N

                                                        35%               N           NH
                                                                              O

                     O            O                                       Guanine (G)
       D   +         C     CH2 O C (CH2)11    CH3
                                                                                      O          O
                                                    D    +    G       +               C    CH2 O C (CH2)11   CH3




                                 CH2CONH
      N
                                                                                           O
                                                                                      HO C (CH2)11    CH3




Mouffouk and Chen (2005)
                   Chemistry of Surfactant Generation –II
                     Ru bipy photo-driven metabolism


                                                                 O

                                           N        Cl       O
                                                         N
                                                Ru
                                           N             N
                          PNA                   N




        Genetic code                     Photochemistry/redox        Fatty acid
                                                centre


Boncella, Bailey, Collis, and Woodruff (2005)
The Pyridyl Fatty Acid System
Overall reaction

                       O         O                       Light                            O                   O
  HS       +   X       C   CH2 O C (CH2)11 CH3                        S   +       X       C       CH3 +    HO C (CH2)11 CH3
                                                    PNA assembly
                   (                      )                                                                (                )




           A       G       T    T   A              G     T       T        A           G           T       T   A        G
       D                          D T                            A    D               C                   A D T        C
           T       C       A    A                  C     A                T                       A


                                      B
                                              O                                                           COO
                                          N                                                                     Ru (PF6)2
                               H2N                COOH                D=                                    3
                                          D                                                   N       N

                                                                 OR
                                   PNA monomer                                                                   CH2CONH
                                                                              N
LA Proto-cell

                  proto-container




  proto-metabolism                  proto-genes

• Organizational closure: cooperation of aggregates
• Thermodynamic and kinetic complementary
  Proto-gene: Peptide Nucleic Acid
              hybridization                                             thermodynamically                       PNA melting in
              of two PNA                                                driven ligation (PACE)                  non-polar solvent
              primers                                                                                           (PACE)




          micelle (lipid interface)
      B                    B                    B                   B                       PNA template directed ligation (PACE)
              O
                  O
                                    O
                                        O
                                                        O
                                                            O
                                                                            O
                                                                                O           PNA-lipid partition (PAs)
          N                     N                   N                   N
H2N                   N                     N                   N                   NH2
                      H                     H                   H
              R                     R                   R                   R




              R=
                                                                                      Peptide Nucleic Acid = PNA
                          Phe           Val         Leu
                                                                                      Like DNA, but peptide backbone
              PNA directed PNA ligation
                                                                                                                                                          CONH
                                                                                                                                                                     2

                                                                                                                                                              O          N


                  C                                                                                                                                       G                  HN
NH
     2
                                                                                                                           NH
                                                                                                                                2
                                                                                                                                             C                                           O                 Sen & Nielsen, 2005
     N        O
                                                                                                                                N        O                                       N
                                                                                                                                                                     O
O
                                                                                                                                                                  G                  HN
     NH                   C                                                                                                O
                                                                                                                                NH                   C                                        O

          N           O
                                                                                                                                     N           O                           O           N

     O
          NH                      C                                                                                             O                                        G                   HN
                                                                                                                                     NH                      C                                    O
                              O                                                                    COOH
                  N
                                                                                                                                             N           O                                    N
                                                                                                       O       N                                                                     O
          O
                  NH                      C                                                        G                                 O
                                                                                                               HN                                                                G                HN
                                                              COOH
                                                                                                                       O
                                                                                                                                             NH                      C                                     O
                          N        O
                                                                  O       N                                                                          N        O
                                                                                                           O       N                                                                     O        N
              O                                           G                   HN
                          NH                  C                                       O                G           H2N                   O                                           G                HN
                                                                                                                                                     NH                  C                                         O

                              N           O                           O        N
                                                                                                                                                         N           O                                     N
                                                                                                                                                                                              O
                                                                                                           EDC
                      O                                           G            H 2N
                                                                                                                                                                                             G                 HN
                               NH                     C                   COOH
                                                                                                                                                 O
                                                                                                                                                          NH                     C                                     O

                                      N           O                           O       N
                                                                                                                                                                 N           O                         O           N

                               O                                          G               HN
                                      NH                      C                                O                                                          O                                       G                    HN
                                                                                                                                                                 NH                      C                                  O

                                              N           O                                N
                                                                                  O
                                                                                                                                                                         N           O                                  N
                                                                                                                                                                                                               O
                                      O                                       G            H2N
                                                                                                                                                                                                                           H2N
                                              NH                  C                                                                                              O                                     G
                                                                                                                                                                         NH                   C
                                                      N       O
                                                                                                                                                                                 N        O
                                                          CONH
                                                                      2
                                                                                                                                                                                     CONH
                                                                                                                                                                                              2
                        Proto-Gene (PNA) experiments
                                                                                                           Sen & Nielsen, 2005


                                                                                                           Template directed
                                                                                                            PNA replication

                                                                                                           Initially 2nd
                                                                                      1
                                                                                            kt
                                                                                                 1         order kinetics
                                                                                   a  x       a



                                                                 (minutes incubation)

                             Conce ntration-depe nde nt PNA ligation k ine tics

        10
         9
         8
Product conc




                                                                                        t:p = 1:2
         7
                                                                                        t:p = 1:3
    (M)




         6
                                                                                        t:p = 1:5
         5
                                                                                        Log. (t:p = 1:2)
         4
         3                                                                              Log. (t:p = 1:3)
         2                                                                              Log. (t:p = 1:5)
         1
         0
               0   20   40            60           80        100         120      140
                                    Incubation time (min)
           PNA solvation & melting in non-polar solvent

                        PNA 58: H-GTAGATCACT-Lys-NH2
                                                                            (Sen & Nielsen, 2005)
                        PNA 60: H-AGTGATCTAC-Lys-NH2
          80

                                                                       Example of
          70
                                                                       PNA solvation
                                                                       and melting
          60
Tm (°C)




                                                                       In DMF (also
                                                                       dioxane)
          50


          40


          30
               0   10    20   30     40   50   60      70   80   90   100
                                   Amount of DMF (%)
     Backbone modified PNAs
                                                           HO        O

                     R5
                                                       T           O
            O
                                           A                             O
                     NH                                                     -                  (Sen & Nielsen, 2005)
                                                                       O P O
                                                                HO       O
                          N            O
                R1

                     O                                         G         O
                          NH                       C                          O
                                                                                       -
                                                                             O P O
                                                                       HO     O
                               N               O
                     R2


                          O                                        C          O

     -(CH2)nCOO-                   NH                      G                        O
                                                                                       -
                                                                                  O P O
     -(CH2)nOH                                         O                     HO     O
R=                                         N
                              R3
     -(CH2)nNH 3+
                                   O                                     A         O
     -(CH2)nCH3                            NH                  T                           O


                                                   N       O
                                                                                           RNA
                               R4
                                                       CONH 2            PNA
                                                           R6
Micellar Electrokinetic Chromatography (MEKC):
        Nonionic Carrier Micelles (Triton)
                                             EP
 PNAA/DNA Duplex
 Unbound DNA
 TX-100 Micelles
                     +                                                                     -
                                               EOF



                     +                                                                     -


                     +                                                                     -

         mdup  K dup M mmic
meff 
            1  K dup M        •Mobility of Triton X-100 is very low
                                 •PNAA/DNA duplexes partition to micelles, reducing meff
                                 •Unbound DNA does not partition, and has a high EP
                                 •Large excess of Triton X-100


                                                                            Schneider (2005)
Proto-gene – II: Peptide




                           2 template strands
   Self replicating peptides


Leucine zipper motif
The SimFit analysis provided an apparent:
catalytic rate constant:
 ka, of 50.6 ± 0.5 M-1.91 s-1
noncatalytic rate constant:
kb, of 5.04 ± 0.03 x 10-4 M-1 s-1.

The catalytic efficiency (ε = ka/kb) of 1.0 x 105
Current status of experiment
 PNA or Peptide Template Selection:

   Library screening

   1) Hunting better sequences as electron relay systems

   2) Finding better candidates for easier ligation (self-replication);

   3) Making it possible for the template to have affinity properties to
      the precursor molecules so that one does not have to work on highly
      concentrated precursor molecules.



Library can be constructed either chemically (PNA) or biologically
(Peptide Phage Display).
Peptide phage display
When libraries of DNA are used as inserts
- libraries of millions of displayed peptides are produced




A shortcut to evolution ! --- functional screening the library
Proto- gene III: Poly aromatic hydrocarbon (PAH)

                              Polycyclic aromatic
                              hydrocarbons

                              … the most abundant
                              molecules in space after
                              molecular hydrogen and
                              carbon monoxide ….

                               Stable against radiation

                              For those species
                              the formation/destruction
                              mechanism is equilibrated
                                Ruiterkamp et al. 2002
       Some examples of possible early templating polymers




Left: the well-known base recognition process                   Ehrenfreund et al. 2005
(a) complementary nucleic bases mediate the polymerization
(b) of a complementary template.

Center: Example of p stacking of particular PAH components
(1) which in term also could polymerize (2) to form a complementary template.

Right: An even simpler PAH p stacking based templating (i) and polymerization (ii)
could occur as PAH elements match complementary PAHs already covalently connected
into a string
Summary: Los Alamos minimal protocell
Uniqueness:                                                    PNA/peptide
• Redefined the notion of a container
   as the metabolic and genetic
   complexes are operating at the
   external interface of a lipid
   aggregate;
                                                    light
•   Genetics, metabolism, and                     sensitizer
    container are chemically and
    functionally integrated for the first
                                                                    lipids
    time: genes part of charge transfer
    process in metabolism;

•   By far the simplest design.
    Rasmussen, Chen, Deamer, Krakauer, Packard,
    Stadler & Bedau, Science 303 (2004) 963
    & Science 305 (2004) 41
   Many possible transitions may exist between nonliving and
     living matter based on our definition of minimal life




                                                                   Rasmussen et al. 2004
                                                                   Ehrenfreund et al. 2005


1) Contemporary life
2) Is it possible to construct different simple living systems, which eventually
   “converge” to life as-we-know-it ?
3) Most of the current published protocell designs could end up satisfying the
   definition of minimal life, but not be able to evolve further. They would then be
   evolutionary blind alleys
4) Finally, it might be possible to create life-forms based on a chemistry and an
   organization that is different enough to life-as-we-know-it, so that truly novel life
   could evolve from it
…we have a few mountains left to climb,
but we will get there.
Acknowledge:
Principal Investigators:                      Postdocs:
                                              Gavin Collis (ANL, C-SIC)
Steen Rasmussen (ANL, EES-6, PACE)            Michael DeClue (ANL, C-SIC)
Jim Bailey (ANL, B-4)                         Dhaval Doshin (ANL, B-4)
Jim Boncella (ANL, C-SIC)                     Chad Knutson (ANL, EES-2)
Gordon Jarvinen (ANL, NMT-DO)                 Fouzi Mouffouk (Rush)
James Maxwell (ANL, ISR-5)                    Pawel Weronski (ANL, T-7 & CNLS)
Pierre-Alain Monnard (ANL, EES-6, PACE)       Xin Zhou (EES-6)
Jim Schneider (CMU)
Andy Shreve (ANL, B-4)                        Goarn Goranovic (Odense, PACE)
                                              Andreea Munteanu (Barcelona, PACE)
Bryan Travis (ANL, EES-2)
                                              Anjana Sen (Copenhagen, PACE)
Woody Woodruff (ANL, C-SIC)
Hans Ziock (ANL, EES-6)
                                              Students:
                                              Camille Attolini (Vienna, PACE)
John S. McCaskill (Ruhr-Universität Bochum)   Gil Banko (Leipzig & Santa Fe Institute)
                                              Harold Fellermann (Barcelona, PACE)
Peter Nielsen (Copenhagen, PACE)
                                              Shane Grosser (CMU)
Martin Nilsson Jacobi (Chalmers, PACE)        Tristan Rocheleau (UCSB - Harvard)
Arvydas Tamulis (Vilnius, PACE)               Sergi Valverde (Barcelona, PACE)
Jerzy Maselko (U Alaska)
Pascale Ehrenfreund (Leiden - JPL NASA)

				
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