Molecular motions inside the cell
A paper in Science this week describes the use of carbon nanotubes to pinpoint the movements of the living cell in fine detail, making for a really nice study in quantitative/mathematical biology.

Noninvasive tracking was accomplished by imaging highly stable near-infrared luminescence of single-walled carbon nanotubes targeted to kinesin-1 motor proteins in COS-7 cells. We observed a regime of active random “stirring” that constitutes an intermediate mode of transport, different from both thermal diffusion and directed motor activity. High-frequency motion was found to be thermally driven. At times greater than 100 milliseconds, nonequilibrium dynamics dominated. In addition to directed transport along microtubules, we observed strong random dynamics driven by myosins that result in enhanced nonspecific transport. We present a quantitative model connecting molecular mechanisms to mesoscopic fluctuations.

The “mesoscopic" scale is more often seen in the context of pure and applied physics (microelectronics, nanofabrication and nanotechnology), though journals such as Soft Matter present research articles giving the same ‘condensed matter’ treatment to biological systems (“Where physics meets chemistry meets biology”).
From ancient Greek μέσος it refers simply to a ‘middle’/intermediate between the molecular and macroscopic scale, where neither atomistic/quantum nor classical physics/bulk models best describe observed behaviour, and novel effects may be described — from interference effects, to quantum confinement (giving rise to band gaps) and charging effects (such as the Coulomb blockade/staircase).
Although often presented as a water-based solvent, the cytosol is more accurately described as a “highly dynamic composite material ” with mechanical properties dominated by microtubules (MTs), F-actin and intermediate filaments; all driven by metabolism-energised polymerisation of actin and tubulin and from motor proteins (specifically nucleotide triphosphate hydrolysis).
The traditional technique to observe cells in motion is fluorescence microscopy, though long-term tracking of single molecules has been hindered by fluorophores’ instabilities and the fluorescence background in cells.
Though biological networks have been termed ‘scale-free’ or ‘-invariant’, and metabolic rate for example is well known to follow a power law, the internal structure of the cell itself is far from self-similar across scales. 

At short times (microseconds to milliseconds), thermal motions should dominate. Between milliseconds and seconds, thermal diffusion might still be relevant, but there is mounting evidence, both in vitro and in vivo, that the motion of larger objects couples to myosin-driven stress fluctuations in the cytoskeleton.

»  Mizuno (2007) Nonequilibrium mechanics of active cytoskeletal networks.
»  Brangwynne (2008) Cytoplasmic diffusion: Molecular motors mix it up.

Here, temporal fluctuations, reminiscent of thermal diffusion in liquids, can arise from nonequilibrium dynamics in the viscoelastic cytoskeleton. On longer time scales, from minutes to hours, directed transport and larger-scale collective motions typically dominate. The motion of probe particles tracked inside cells has been classified as subdiffusive, diffusive, or superdiffusive. Such classifications, however, obscure the distinction between thermally driven and nonequilibrium fluctuations and are inadequate to identify intracellular material properties.

Motor proteins direct a whole host of molecular motions, kinesins and myosins being among the most heavily studied in vitro. Using fluorescence microscopy to track individual motor proteins is not only limited by instability of fluorophores, but the quality of the images taken (“signal to noise”) and efficiency of targetting probes to specific molecules.
Modern optical equipment and carefully designed fluorescent dyes have enabled experiments tracking single molecules at a time, though in living cells the authors note these experiments’ timeframes have been limited to around a second.

Their solution was to use single-walled carbon nanotubes (SWNTs), “stiff quasi–one-dimensional tubular all-carbon nanostructures with diameters of ~1 nm and persistence lengths above 10 μm” — which have the convenient property of luminescence in the near-infrared, a region ‘virtually free of autofluorescence in biological tissues’. Not only this, but the excitation time is ~100ps, such that high excitation can give ~1 ms resolution (1 ms = 109 ps).
The nanotubes were dispersed throughout the cell wrapped in short DNA oligonucleotides, with HaloTag protein fusion tags covalently attaching them specifically onto kinesin motor proteins (see Fig. 1, above).

Besides observing directed kinesin-driven transport on MTs, it is possible to directly observe fluctuations of the MT network because a moving kinesin must be bound to a MT. The MT tracks are embedded in the viscoelastic actin cytoskeleton, which in turn fluctuates as a result of stresses generated by cytoplasmic myosins.


With just 100 per cell, the group could track kinesin for up to an hour and a half, observing ~30% of them moving with some sense of direction; the rest locally constrained and moving in a random (stochastic) manner.
Some of the kinesins moved the whole length of the cell, suggesting they had cargo vesicles [along with other motor proteins] attached. Calculating mean squared displacement, MSD, of the molecules’ trajectories showed it grew over time following a power law which could be used to characterise the motion, 〈Δr2(τ)〉∝ τα(where r is distance travelled in the focal plane and τ the lag time). The exponent α shifted from ¼ to 1 between 5 ms and 2.5 s, indicating clear scale variance to the motion.
After this, the group acquired the nanotubes’ fluorescent signal at a rate of four frames per second ‒ using this 250 ms window to observe an intermediate between the thermal diffusion seen on the short timescales and directed motor activity.
With a well-designed control or two, they showed that the transverse motion of the nanotube-marked microtubules was not due to kinesin motors, but reflecting intrinsic dynamics of the cytoskeleton.

The way the relatively rigid MTs report these dynamics depends on two restoring forces: the elastic force of bent MTs and the force exerted by the strained cytoskeletal matrix in which the MTs are embedded. Because it is hard to bend an elastic rod on short length scales, the surrounding matrix yields to the MT when it is deformed on short length scales. By contrast, the MT yields to matrix forces for deflections of wavelength larger than ~1 μm. The shorter-wavelength MT deflections relax faster than our 5-ms frame rate. Therefore, we assume that the transverse MT motion we observe reflects the (active or passive) strain fluctuations of the surrounding matrix.
The MSD power-law exponent α generally reflects the randomness of motion. More precisely, in any medium, the MSD of an embedded probe particle is governed both by the material properties of the medium and the temporal characteristics of the forces driving the particle. For thermally driven Brownian motion in simple liquids, the MSD exponent α = 1. For thermal motion in viscoelastic media, which exhibit time- and frequency-dependent viscosity and elasticity, α < 1 strictly holds. For viscoelastic materials, the stiffness G(ω) typically increases with a power of frequency ω: G(ω) ∝ ωβ. This is observed in polymer solutions, where the viscoelastic exponent β ≈ 0.5 to 0.8, as well as in cells, where β ≈ 0.1 to 0.2 on time scales on the order of seconds. This value of the exponent is close to what is expected for purely elastic materials, where β = 0. 
The nearly elastic behavior of cells can be understood as a consequence of strong cross-linking in the cytoskeleton.
 Knowing the driving forces, it is possible to construct a relation between MSD exponent α and viscoelastic exponent β. For thermal driving forces, the MSD exponent α = β. Thermal fluctuations can therefore never appear as “superdiffusive” motion with α > 1. Nonthermal driving, by contrast, can result in superdiffusive motion. Theory provides a specific prediction for motion in nearly elastic solids driven by random stress fluctuations with long correlation times and sudden transitions: α = 1 + 2β. This prediction is expected to apply for cytoskeletal stress fluctuations caused by randomly distributed cytoplasmic myosin minifilaments. Myosin locally contracts the actin network with an attachment time of several seconds, followed by sudden release. Some hints of this predicted scaling have been reported for cells and reconstituted acto-myosin model systems. When β = 0 (i.e., in the elastic limit), the resulting MSDs can look deceptively like Brownian motion in a simple liquid, although the physical reason is entirely different. For observation times τ longer than the correlation time of the driving forces, the MSD is predicted to level off, as we observed. In our experiments, the stress correlation time should correspond to typical cytoplasmic myosin motor engagement times, which are indeed reported to be ~10 s in cells.

Still attached to microtubules, the kinesin molecules exhibit vigorous random (Brownian-like) motion as they are buffeted by myosins as described ‒ likely thrusting MTs into the path of other cellular particles. Tubulin forms strong tubular filaments embedded in a more flexible actin network. Nonmuscle myosin II exerts mechanical stress on it, which is released ‘suddenly’ as random stirring of the whole filament network, including the microtubules.

We observed a transition between thermal dynamics in the dominantly elastic cytoskeleton at short times to strongly nonequilibrium power-law dynamics, likely driven by myosin activity, at intermediate times. When the time exceeded the correlation time of the random stress generators, the intermediate regime was followed by a saturation to a maximum MSD, nearly constant over time. Note that in this regime, the MSD amplitude corresponds to a root mean square displacement of ~500 nm, which is larger than the estimated mesh size of the actin network, and thus larger than the expected spacing of obstacles in the crowded cytoplasm.

The authors lastly used myosin inhibitor blebbistatin to block myosin from the actin network, confirming their hypothesis with a dose-dependent reduction in what they call the amplitude of active stirring, and exponent α, “establishing nonmuscle myosin II as the dominant driving factor for random cytoskeletal stirring”.

We can explain the regimes we observe by a quantitative model of cytoskeletal fluctuations and directed motor motion that describes the transition from thermal motion to nonequilibrium stirring dynamics driven by myosin, as well as the transition from stirring dynamics to directed transport driven by kinesin. Our observations were made possible by the use of SWNT labels for broadband molecular tracking in cells. Many questions concerning motor transport in cells will now be addressable using this approach. We have focused here on the stirring dynamics, which constitute an important mode of active intracellular transport between the limits of random thermal diffusion and directed transport, accelerating nonspecific transport through the nanoporous cytoskeleton.

Lead author Nikta Fakhri will soon leave the Göttingen Institute for Biophysics to join the faculty at MIT as assistant professor of physics. Fakhri gave a talk in Massachussets last year on the topic, to the Chemical Engineering department in which some of the details of this paper made their debut:

The discovery of fullerenes provided exciting insights into how highly symmetrical structures of pure carbon can have remarkable physical properties. Single-walled carbon nanotubes (SWNTs) are the vanguard of such architectures. The organization of the hexagonal honeycomb carbon lattice into high-aspect-ratio cylinders with a variety of helical symmetries creates very unusual macromolecular structures representing an emerging research area in condensed matter physics and materials science: traditionally hard materials appearing in new soft matter applications and environments.
… the dynamics of SWNTs in liquids are essentially polymer-like. By exploiting the intrinsic near-infrared fluorescence of semiconducting SWNTs, we have imaged the Brownian motion of individual nanotubes in water and have measured directly the bending stiffness of SWNTs. The semiflexible chain model represents accurately the configurational dynamics of SWNTs suspended in water. Interestingly, the persistence length of SWNTs is comparable to that of biopolymers. This finding paves the way for using SWNTs as a model system for semiflexible polymers to answer long-standing fundamental questions in polymer physics.
… the confined dynamics of stiff macromolecules in crowded environments [are] a common feature of polymer composites and the cell cytoskeleton. In fixed porous networks, we find that even a small bending flexibility strongly enhances SWNTs’ motion. This ends a 30-year-old debate in polymer physics: the rotational diffusion constant is proportional to the filament bending compliance and counter-intuitively, independent of the network porosity. The dynamics of SWNTs in equilibrium and non-equilibrium biopolymer networks is more complex.
At long times, SWNTs reptate in the networks. At short times SWNTs can sample the spectrum of local stresses in equilibrium networks. In the non-equilibrium networks we observe strong local shape fluctuations driven by force generating molecular motors. I will discuss a newly developed microrheology technique in which we use nanotubes as “stealth probes” to measure viscoelastic properties of the host media. Finally, I will introduce a new single-molecule technique based on ultra-stable near-infrared fluorescence of short SWNTs, to study intracellular transport dynamics in living cells and in whole organisms. The combination of long-time stability and high signal-to-noise ratio enables the accurate long-term tracking of single motor proteins tagged with SWNTs traversing the entire cell. Remarkably, we can distinguish the motor protein’s motion along its microtubule track from the track’s underlying random non-thermal fluctuations.

She envisions the technology as applicable beyond probing biophysical questions, in the design of 'active' technical materials.
“Imagine a microscopic biomedical device that mixes tiny samples of blood with reagents to detect disease or smart filters that separate squishy from rigid materials.”
Fakhri will join the Physics of Living Systems group, seemingly on such a bio-materials science project. MIT lab colleague Jeremy England, known for work showing that E. coli reproduction is close to thermodynamic limits of efficiency, spoke of common interest in the cytosol and diffusive processes.

“We’re interested in the non-equilibrium thermodynamics of biological organization, so that could be construed to be about evolution and the origins of life or just about how you make or design self-replicators with desired properties.”
“Increasingly there are now instruments where you can make quantitative measurements on fluorescently labeled proteins in live cells,” England explains. “The cell biologists have their language and their frame of analysis that they’re most comfortable with for describing the phenomenon, but if there are interesting phenomena that are only going to be identifiable if you do the right quantitative analysis on all these numbers that you can now measure in the cell, then it’s useful to have people who are a bit more theoretically minded or physics minded who are there, when rubber meets road, when the data is being generated and helping to influence what kind of experiments get done.”
“We’re looking, for example, at diffusion of proteins in cells. Diffusion as a qualitative phenomenon is just things spreading out over space, but as a quantitative phenomenon, you can look at things like how rapidly a protein that’s labeled over here in the cell will wander over to another region of the cell that’s a certain distance away, and if you can make measurements of that, then you can start to say things that are more specific about characteristics of the diffusion that you are observing than simply seeing it spread out. And in those quantitative measurements, you can sometimes then see differences perhaps between different cells, or different conditions for the same type of cell, that may have biological relevance but that you wouldn’t have necessarily identified without the quantitative analysis,” England says.

⌇  Fakhri et al. (2014) High-resolution mapping of intracellular fluctuations using carbon nanotubes. Science, 344(1687), 1031-5
See also:⌇  Levine and MacKintosh (2009) The mechanics and fluctuation spectrum of active gels. J Phys Chem B, 113, 3820–3830⌇  MacKintosh and Levine (2008) Nonequilibrium mechanics and dynamics of motor-activated gels. Phys Rev Lett, 100, 018104⌇  Lau et al. (2003) Microrheology, stress fluctuations, and active behavior of living cells. Phys Rev Lett, 91, 198101⇢  Related post : water’s SED failure in molecular orientational diffusion
Molecular motions inside the cell
A paper in Science this week describes the use of carbon nanotubes to pinpoint the movements of the living cell in fine detail, making for a really nice study in quantitative/mathematical biology.

Noninvasive tracking was accomplished by imaging highly stable near-infrared luminescence of single-walled carbon nanotubes targeted to kinesin-1 motor proteins in COS-7 cells. We observed a regime of active random “stirring” that constitutes an intermediate mode of transport, different from both thermal diffusion and directed motor activity. High-frequency motion was found to be thermally driven. At times greater than 100 milliseconds, nonequilibrium dynamics dominated. In addition to directed transport along microtubules, we observed strong random dynamics driven by myosins that result in enhanced nonspecific transport. We present a quantitative model connecting molecular mechanisms to mesoscopic fluctuations.

The “mesoscopic" scale is more often seen in the context of pure and applied physics (microelectronics, nanofabrication and nanotechnology), though journals such as Soft Matter present research articles giving the same ‘condensed matter’ treatment to biological systems (“Where physics meets chemistry meets biology”).
From ancient Greek μέσος it refers simply to a ‘middle’/intermediate between the molecular and macroscopic scale, where neither atomistic/quantum nor classical physics/bulk models best describe observed behaviour, and novel effects may be described — from interference effects, to quantum confinement (giving rise to band gaps) and charging effects (such as the Coulomb blockade/staircase).
Although often presented as a water-based solvent, the cytosol is more accurately described as a “highly dynamic composite material ” with mechanical properties dominated by microtubules (MTs), F-actin and intermediate filaments; all driven by metabolism-energised polymerisation of actin and tubulin and from motor proteins (specifically nucleotide triphosphate hydrolysis).
The traditional technique to observe cells in motion is fluorescence microscopy, though long-term tracking of single molecules has been hindered by fluorophores’ instabilities and the fluorescence background in cells.
Though biological networks have been termed ‘scale-free’ or ‘-invariant’, and metabolic rate for example is well known to follow a power law, the internal structure of the cell itself is far from self-similar across scales. 

At short times (microseconds to milliseconds), thermal motions should dominate. Between milliseconds and seconds, thermal diffusion might still be relevant, but there is mounting evidence, both in vitro and in vivo, that the motion of larger objects couples to myosin-driven stress fluctuations in the cytoskeleton.

»  Mizuno (2007) Nonequilibrium mechanics of active cytoskeletal networks.
»  Brangwynne (2008) Cytoplasmic diffusion: Molecular motors mix it up.

Here, temporal fluctuations, reminiscent of thermal diffusion in liquids, can arise from nonequilibrium dynamics in the viscoelastic cytoskeleton. On longer time scales, from minutes to hours, directed transport and larger-scale collective motions typically dominate. The motion of probe particles tracked inside cells has been classified as subdiffusive, diffusive, or superdiffusive. Such classifications, however, obscure the distinction between thermally driven and nonequilibrium fluctuations and are inadequate to identify intracellular material properties.

Motor proteins direct a whole host of molecular motions, kinesins and myosins being among the most heavily studied in vitro. Using fluorescence microscopy to track individual motor proteins is not only limited by instability of fluorophores, but the quality of the images taken (“signal to noise”) and efficiency of targetting probes to specific molecules.
Modern optical equipment and carefully designed fluorescent dyes have enabled experiments tracking single molecules at a time, though in living cells the authors note these experiments’ timeframes have been limited to around a second.

Their solution was to use single-walled carbon nanotubes (SWNTs), “stiff quasi–one-dimensional tubular all-carbon nanostructures with diameters of ~1 nm and persistence lengths above 10 μm” — which have the convenient property of luminescence in the near-infrared, a region ‘virtually free of autofluorescence in biological tissues’. Not only this, but the excitation time is ~100ps, such that high excitation can give ~1 ms resolution (1 ms = 109 ps).
The nanotubes were dispersed throughout the cell wrapped in short DNA oligonucleotides, with HaloTag protein fusion tags covalently attaching them specifically onto kinesin motor proteins (see Fig. 1, above).

Besides observing directed kinesin-driven transport on MTs, it is possible to directly observe fluctuations of the MT network because a moving kinesin must be bound to a MT. The MT tracks are embedded in the viscoelastic actin cytoskeleton, which in turn fluctuates as a result of stresses generated by cytoplasmic myosins.


With just 100 per cell, the group could track kinesin for up to an hour and a half, observing ~30% of them moving with some sense of direction; the rest locally constrained and moving in a random (stochastic) manner.
Some of the kinesins moved the whole length of the cell, suggesting they had cargo vesicles [along with other motor proteins] attached. Calculating mean squared displacement, MSD, of the molecules’ trajectories showed it grew over time following a power law which could be used to characterise the motion, 〈Δr2(τ)〉∝ τα(where r is distance travelled in the focal plane and τ the lag time). The exponent α shifted from ¼ to 1 between 5 ms and 2.5 s, indicating clear scale variance to the motion.
After this, the group acquired the nanotubes’ fluorescent signal at a rate of four frames per second ‒ using this 250 ms window to observe an intermediate between the thermal diffusion seen on the short timescales and directed motor activity.
With a well-designed control or two, they showed that the transverse motion of the nanotube-marked microtubules was not due to kinesin motors, but reflecting intrinsic dynamics of the cytoskeleton.

The way the relatively rigid MTs report these dynamics depends on two restoring forces: the elastic force of bent MTs and the force exerted by the strained cytoskeletal matrix in which the MTs are embedded. Because it is hard to bend an elastic rod on short length scales, the surrounding matrix yields to the MT when it is deformed on short length scales. By contrast, the MT yields to matrix forces for deflections of wavelength larger than ~1 μm. The shorter-wavelength MT deflections relax faster than our 5-ms frame rate. Therefore, we assume that the transverse MT motion we observe reflects the (active or passive) strain fluctuations of the surrounding matrix.
The MSD power-law exponent α generally reflects the randomness of motion. More precisely, in any medium, the MSD of an embedded probe particle is governed both by the material properties of the medium and the temporal characteristics of the forces driving the particle. For thermally driven Brownian motion in simple liquids, the MSD exponent α = 1. For thermal motion in viscoelastic media, which exhibit time- and frequency-dependent viscosity and elasticity, α < 1 strictly holds. For viscoelastic materials, the stiffness G(ω) typically increases with a power of frequency ω: G(ω) ∝ ωβ. This is observed in polymer solutions, where the viscoelastic exponent β ≈ 0.5 to 0.8, as well as in cells, where β ≈ 0.1 to 0.2 on time scales on the order of seconds. This value of the exponent is close to what is expected for purely elastic materials, where β = 0. 
The nearly elastic behavior of cells can be understood as a consequence of strong cross-linking in the cytoskeleton.
 Knowing the driving forces, it is possible to construct a relation between MSD exponent α and viscoelastic exponent β. For thermal driving forces, the MSD exponent α = β. Thermal fluctuations can therefore never appear as “superdiffusive” motion with α > 1. Nonthermal driving, by contrast, can result in superdiffusive motion. Theory provides a specific prediction for motion in nearly elastic solids driven by random stress fluctuations with long correlation times and sudden transitions: α = 1 + 2β. This prediction is expected to apply for cytoskeletal stress fluctuations caused by randomly distributed cytoplasmic myosin minifilaments. Myosin locally contracts the actin network with an attachment time of several seconds, followed by sudden release. Some hints of this predicted scaling have been reported for cells and reconstituted acto-myosin model systems. When β = 0 (i.e., in the elastic limit), the resulting MSDs can look deceptively like Brownian motion in a simple liquid, although the physical reason is entirely different. For observation times τ longer than the correlation time of the driving forces, the MSD is predicted to level off, as we observed. In our experiments, the stress correlation time should correspond to typical cytoplasmic myosin motor engagement times, which are indeed reported to be ~10 s in cells.

Still attached to microtubules, the kinesin molecules exhibit vigorous random (Brownian-like) motion as they are buffeted by myosins as described ‒ likely thrusting MTs into the path of other cellular particles. Tubulin forms strong tubular filaments embedded in a more flexible actin network. Nonmuscle myosin II exerts mechanical stress on it, which is released ‘suddenly’ as random stirring of the whole filament network, including the microtubules.

We observed a transition between thermal dynamics in the dominantly elastic cytoskeleton at short times to strongly nonequilibrium power-law dynamics, likely driven by myosin activity, at intermediate times. When the time exceeded the correlation time of the random stress generators, the intermediate regime was followed by a saturation to a maximum MSD, nearly constant over time. Note that in this regime, the MSD amplitude corresponds to a root mean square displacement of ~500 nm, which is larger than the estimated mesh size of the actin network, and thus larger than the expected spacing of obstacles in the crowded cytoplasm.

The authors lastly used myosin inhibitor blebbistatin to block myosin from the actin network, confirming their hypothesis with a dose-dependent reduction in what they call the amplitude of active stirring, and exponent α, “establishing nonmuscle myosin II as the dominant driving factor for random cytoskeletal stirring”.

We can explain the regimes we observe by a quantitative model of cytoskeletal fluctuations and directed motor motion that describes the transition from thermal motion to nonequilibrium stirring dynamics driven by myosin, as well as the transition from stirring dynamics to directed transport driven by kinesin. Our observations were made possible by the use of SWNT labels for broadband molecular tracking in cells. Many questions concerning motor transport in cells will now be addressable using this approach. We have focused here on the stirring dynamics, which constitute an important mode of active intracellular transport between the limits of random thermal diffusion and directed transport, accelerating nonspecific transport through the nanoporous cytoskeleton.

Lead author Nikta Fakhri will soon leave the Göttingen Institute for Biophysics to join the faculty at MIT as assistant professor of physics. Fakhri gave a talk in Massachussets last year on the topic, to the Chemical Engineering department in which some of the details of this paper made their debut:

The discovery of fullerenes provided exciting insights into how highly symmetrical structures of pure carbon can have remarkable physical properties. Single-walled carbon nanotubes (SWNTs) are the vanguard of such architectures. The organization of the hexagonal honeycomb carbon lattice into high-aspect-ratio cylinders with a variety of helical symmetries creates very unusual macromolecular structures representing an emerging research area in condensed matter physics and materials science: traditionally hard materials appearing in new soft matter applications and environments.
… the dynamics of SWNTs in liquids are essentially polymer-like. By exploiting the intrinsic near-infrared fluorescence of semiconducting SWNTs, we have imaged the Brownian motion of individual nanotubes in water and have measured directly the bending stiffness of SWNTs. The semiflexible chain model represents accurately the configurational dynamics of SWNTs suspended in water. Interestingly, the persistence length of SWNTs is comparable to that of biopolymers. This finding paves the way for using SWNTs as a model system for semiflexible polymers to answer long-standing fundamental questions in polymer physics.
… the confined dynamics of stiff macromolecules in crowded environments [are] a common feature of polymer composites and the cell cytoskeleton. In fixed porous networks, we find that even a small bending flexibility strongly enhances SWNTs’ motion. This ends a 30-year-old debate in polymer physics: the rotational diffusion constant is proportional to the filament bending compliance and counter-intuitively, independent of the network porosity. The dynamics of SWNTs in equilibrium and non-equilibrium biopolymer networks is more complex.
At long times, SWNTs reptate in the networks. At short times SWNTs can sample the spectrum of local stresses in equilibrium networks. In the non-equilibrium networks we observe strong local shape fluctuations driven by force generating molecular motors. I will discuss a newly developed microrheology technique in which we use nanotubes as “stealth probes” to measure viscoelastic properties of the host media. Finally, I will introduce a new single-molecule technique based on ultra-stable near-infrared fluorescence of short SWNTs, to study intracellular transport dynamics in living cells and in whole organisms. The combination of long-time stability and high signal-to-noise ratio enables the accurate long-term tracking of single motor proteins tagged with SWNTs traversing the entire cell. Remarkably, we can distinguish the motor protein’s motion along its microtubule track from the track’s underlying random non-thermal fluctuations.

She envisions the technology as applicable beyond probing biophysical questions, in the design of 'active' technical materials.
“Imagine a microscopic biomedical device that mixes tiny samples of blood with reagents to detect disease or smart filters that separate squishy from rigid materials.”
Fakhri will join the Physics of Living Systems group, seemingly on such a bio-materials science project. MIT lab colleague Jeremy England, known for work showing that E. coli reproduction is close to thermodynamic limits of efficiency, spoke of common interest in the cytosol and diffusive processes.

“We’re interested in the non-equilibrium thermodynamics of biological organization, so that could be construed to be about evolution and the origins of life or just about how you make or design self-replicators with desired properties.”
“Increasingly there are now instruments where you can make quantitative measurements on fluorescently labeled proteins in live cells,” England explains. “The cell biologists have their language and their frame of analysis that they’re most comfortable with for describing the phenomenon, but if there are interesting phenomena that are only going to be identifiable if you do the right quantitative analysis on all these numbers that you can now measure in the cell, then it’s useful to have people who are a bit more theoretically minded or physics minded who are there, when rubber meets road, when the data is being generated and helping to influence what kind of experiments get done.”
“We’re looking, for example, at diffusion of proteins in cells. Diffusion as a qualitative phenomenon is just things spreading out over space, but as a quantitative phenomenon, you can look at things like how rapidly a protein that’s labeled over here in the cell will wander over to another region of the cell that’s a certain distance away, and if you can make measurements of that, then you can start to say things that are more specific about characteristics of the diffusion that you are observing than simply seeing it spread out. And in those quantitative measurements, you can sometimes then see differences perhaps between different cells, or different conditions for the same type of cell, that may have biological relevance but that you wouldn’t have necessarily identified without the quantitative analysis,” England says.

⌇  Fakhri et al. (2014) High-resolution mapping of intracellular fluctuations using carbon nanotubes. Science, 344(1687), 1031-5
See also:⌇  Levine and MacKintosh (2009) The mechanics and fluctuation spectrum of active gels. J Phys Chem B, 113, 3820–3830⌇  MacKintosh and Levine (2008) Nonequilibrium mechanics and dynamics of motor-activated gels. Phys Rev Lett, 100, 018104⌇  Lau et al. (2003) Microrheology, stress fluctuations, and active behavior of living cells. Phys Rev Lett, 91, 198101⇢  Related post : water’s SED failure in molecular orientational diffusion
Molecular motions inside the cell
A paper in Science this week describes the use of carbon nanotubes to pinpoint the movements of the living cell in fine detail, making for a really nice study in quantitative/mathematical biology.

Noninvasive tracking was accomplished by imaging highly stable near-infrared luminescence of single-walled carbon nanotubes targeted to kinesin-1 motor proteins in COS-7 cells. We observed a regime of active random “stirring” that constitutes an intermediate mode of transport, different from both thermal diffusion and directed motor activity. High-frequency motion was found to be thermally driven. At times greater than 100 milliseconds, nonequilibrium dynamics dominated. In addition to directed transport along microtubules, we observed strong random dynamics driven by myosins that result in enhanced nonspecific transport. We present a quantitative model connecting molecular mechanisms to mesoscopic fluctuations.

The “mesoscopic" scale is more often seen in the context of pure and applied physics (microelectronics, nanofabrication and nanotechnology), though journals such as Soft Matter present research articles giving the same ‘condensed matter’ treatment to biological systems (“Where physics meets chemistry meets biology”).
From ancient Greek μέσος it refers simply to a ‘middle’/intermediate between the molecular and macroscopic scale, where neither atomistic/quantum nor classical physics/bulk models best describe observed behaviour, and novel effects may be described — from interference effects, to quantum confinement (giving rise to band gaps) and charging effects (such as the Coulomb blockade/staircase).
Although often presented as a water-based solvent, the cytosol is more accurately described as a “highly dynamic composite material ” with mechanical properties dominated by microtubules (MTs), F-actin and intermediate filaments; all driven by metabolism-energised polymerisation of actin and tubulin and from motor proteins (specifically nucleotide triphosphate hydrolysis).
The traditional technique to observe cells in motion is fluorescence microscopy, though long-term tracking of single molecules has been hindered by fluorophores’ instabilities and the fluorescence background in cells.
Though biological networks have been termed ‘scale-free’ or ‘-invariant’, and metabolic rate for example is well known to follow a power law, the internal structure of the cell itself is far from self-similar across scales. 

At short times (microseconds to milliseconds), thermal motions should dominate. Between milliseconds and seconds, thermal diffusion might still be relevant, but there is mounting evidence, both in vitro and in vivo, that the motion of larger objects couples to myosin-driven stress fluctuations in the cytoskeleton.

»  Mizuno (2007) Nonequilibrium mechanics of active cytoskeletal networks.
»  Brangwynne (2008) Cytoplasmic diffusion: Molecular motors mix it up.

Here, temporal fluctuations, reminiscent of thermal diffusion in liquids, can arise from nonequilibrium dynamics in the viscoelastic cytoskeleton. On longer time scales, from minutes to hours, directed transport and larger-scale collective motions typically dominate. The motion of probe particles tracked inside cells has been classified as subdiffusive, diffusive, or superdiffusive. Such classifications, however, obscure the distinction between thermally driven and nonequilibrium fluctuations and are inadequate to identify intracellular material properties.

Motor proteins direct a whole host of molecular motions, kinesins and myosins being among the most heavily studied in vitro. Using fluorescence microscopy to track individual motor proteins is not only limited by instability of fluorophores, but the quality of the images taken (“signal to noise”) and efficiency of targetting probes to specific molecules.
Modern optical equipment and carefully designed fluorescent dyes have enabled experiments tracking single molecules at a time, though in living cells the authors note these experiments’ timeframes have been limited to around a second.

Their solution was to use single-walled carbon nanotubes (SWNTs), “stiff quasi–one-dimensional tubular all-carbon nanostructures with diameters of ~1 nm and persistence lengths above 10 μm” — which have the convenient property of luminescence in the near-infrared, a region ‘virtually free of autofluorescence in biological tissues’. Not only this, but the excitation time is ~100ps, such that high excitation can give ~1 ms resolution (1 ms = 109 ps).
The nanotubes were dispersed throughout the cell wrapped in short DNA oligonucleotides, with HaloTag protein fusion tags covalently attaching them specifically onto kinesin motor proteins (see Fig. 1, above).

Besides observing directed kinesin-driven transport on MTs, it is possible to directly observe fluctuations of the MT network because a moving kinesin must be bound to a MT. The MT tracks are embedded in the viscoelastic actin cytoskeleton, which in turn fluctuates as a result of stresses generated by cytoplasmic myosins.


With just 100 per cell, the group could track kinesin for up to an hour and a half, observing ~30% of them moving with some sense of direction; the rest locally constrained and moving in a random (stochastic) manner.
Some of the kinesins moved the whole length of the cell, suggesting they had cargo vesicles [along with other motor proteins] attached. Calculating mean squared displacement, MSD, of the molecules’ trajectories showed it grew over time following a power law which could be used to characterise the motion, 〈Δr2(τ)〉∝ τα(where r is distance travelled in the focal plane and τ the lag time). The exponent α shifted from ¼ to 1 between 5 ms and 2.5 s, indicating clear scale variance to the motion.
After this, the group acquired the nanotubes’ fluorescent signal at a rate of four frames per second ‒ using this 250 ms window to observe an intermediate between the thermal diffusion seen on the short timescales and directed motor activity.
With a well-designed control or two, they showed that the transverse motion of the nanotube-marked microtubules was not due to kinesin motors, but reflecting intrinsic dynamics of the cytoskeleton.

The way the relatively rigid MTs report these dynamics depends on two restoring forces: the elastic force of bent MTs and the force exerted by the strained cytoskeletal matrix in which the MTs are embedded. Because it is hard to bend an elastic rod on short length scales, the surrounding matrix yields to the MT when it is deformed on short length scales. By contrast, the MT yields to matrix forces for deflections of wavelength larger than ~1 μm. The shorter-wavelength MT deflections relax faster than our 5-ms frame rate. Therefore, we assume that the transverse MT motion we observe reflects the (active or passive) strain fluctuations of the surrounding matrix.
The MSD power-law exponent α generally reflects the randomness of motion. More precisely, in any medium, the MSD of an embedded probe particle is governed both by the material properties of the medium and the temporal characteristics of the forces driving the particle. For thermally driven Brownian motion in simple liquids, the MSD exponent α = 1. For thermal motion in viscoelastic media, which exhibit time- and frequency-dependent viscosity and elasticity, α < 1 strictly holds. For viscoelastic materials, the stiffness G(ω) typically increases with a power of frequency ω: G(ω) ∝ ωβ. This is observed in polymer solutions, where the viscoelastic exponent β ≈ 0.5 to 0.8, as well as in cells, where β ≈ 0.1 to 0.2 on time scales on the order of seconds. This value of the exponent is close to what is expected for purely elastic materials, where β = 0. 
The nearly elastic behavior of cells can be understood as a consequence of strong cross-linking in the cytoskeleton.
 Knowing the driving forces, it is possible to construct a relation between MSD exponent α and viscoelastic exponent β. For thermal driving forces, the MSD exponent α = β. Thermal fluctuations can therefore never appear as “superdiffusive” motion with α > 1. Nonthermal driving, by contrast, can result in superdiffusive motion. Theory provides a specific prediction for motion in nearly elastic solids driven by random stress fluctuations with long correlation times and sudden transitions: α = 1 + 2β. This prediction is expected to apply for cytoskeletal stress fluctuations caused by randomly distributed cytoplasmic myosin minifilaments. Myosin locally contracts the actin network with an attachment time of several seconds, followed by sudden release. Some hints of this predicted scaling have been reported for cells and reconstituted acto-myosin model systems. When β = 0 (i.e., in the elastic limit), the resulting MSDs can look deceptively like Brownian motion in a simple liquid, although the physical reason is entirely different. For observation times τ longer than the correlation time of the driving forces, the MSD is predicted to level off, as we observed. In our experiments, the stress correlation time should correspond to typical cytoplasmic myosin motor engagement times, which are indeed reported to be ~10 s in cells.

Still attached to microtubules, the kinesin molecules exhibit vigorous random (Brownian-like) motion as they are buffeted by myosins as described ‒ likely thrusting MTs into the path of other cellular particles. Tubulin forms strong tubular filaments embedded in a more flexible actin network. Nonmuscle myosin II exerts mechanical stress on it, which is released ‘suddenly’ as random stirring of the whole filament network, including the microtubules.

We observed a transition between thermal dynamics in the dominantly elastic cytoskeleton at short times to strongly nonequilibrium power-law dynamics, likely driven by myosin activity, at intermediate times. When the time exceeded the correlation time of the random stress generators, the intermediate regime was followed by a saturation to a maximum MSD, nearly constant over time. Note that in this regime, the MSD amplitude corresponds to a root mean square displacement of ~500 nm, which is larger than the estimated mesh size of the actin network, and thus larger than the expected spacing of obstacles in the crowded cytoplasm.

The authors lastly used myosin inhibitor blebbistatin to block myosin from the actin network, confirming their hypothesis with a dose-dependent reduction in what they call the amplitude of active stirring, and exponent α, “establishing nonmuscle myosin II as the dominant driving factor for random cytoskeletal stirring”.

We can explain the regimes we observe by a quantitative model of cytoskeletal fluctuations and directed motor motion that describes the transition from thermal motion to nonequilibrium stirring dynamics driven by myosin, as well as the transition from stirring dynamics to directed transport driven by kinesin. Our observations were made possible by the use of SWNT labels for broadband molecular tracking in cells. Many questions concerning motor transport in cells will now be addressable using this approach. We have focused here on the stirring dynamics, which constitute an important mode of active intracellular transport between the limits of random thermal diffusion and directed transport, accelerating nonspecific transport through the nanoporous cytoskeleton.

Lead author Nikta Fakhri will soon leave the Göttingen Institute for Biophysics to join the faculty at MIT as assistant professor of physics. Fakhri gave a talk in Massachussets last year on the topic, to the Chemical Engineering department in which some of the details of this paper made their debut:

The discovery of fullerenes provided exciting insights into how highly symmetrical structures of pure carbon can have remarkable physical properties. Single-walled carbon nanotubes (SWNTs) are the vanguard of such architectures. The organization of the hexagonal honeycomb carbon lattice into high-aspect-ratio cylinders with a variety of helical symmetries creates very unusual macromolecular structures representing an emerging research area in condensed matter physics and materials science: traditionally hard materials appearing in new soft matter applications and environments.
… the dynamics of SWNTs in liquids are essentially polymer-like. By exploiting the intrinsic near-infrared fluorescence of semiconducting SWNTs, we have imaged the Brownian motion of individual nanotubes in water and have measured directly the bending stiffness of SWNTs. The semiflexible chain model represents accurately the configurational dynamics of SWNTs suspended in water. Interestingly, the persistence length of SWNTs is comparable to that of biopolymers. This finding paves the way for using SWNTs as a model system for semiflexible polymers to answer long-standing fundamental questions in polymer physics.
… the confined dynamics of stiff macromolecules in crowded environments [are] a common feature of polymer composites and the cell cytoskeleton. In fixed porous networks, we find that even a small bending flexibility strongly enhances SWNTs’ motion. This ends a 30-year-old debate in polymer physics: the rotational diffusion constant is proportional to the filament bending compliance and counter-intuitively, independent of the network porosity. The dynamics of SWNTs in equilibrium and non-equilibrium biopolymer networks is more complex.
At long times, SWNTs reptate in the networks. At short times SWNTs can sample the spectrum of local stresses in equilibrium networks. In the non-equilibrium networks we observe strong local shape fluctuations driven by force generating molecular motors. I will discuss a newly developed microrheology technique in which we use nanotubes as “stealth probes” to measure viscoelastic properties of the host media. Finally, I will introduce a new single-molecule technique based on ultra-stable near-infrared fluorescence of short SWNTs, to study intracellular transport dynamics in living cells and in whole organisms. The combination of long-time stability and high signal-to-noise ratio enables the accurate long-term tracking of single motor proteins tagged with SWNTs traversing the entire cell. Remarkably, we can distinguish the motor protein’s motion along its microtubule track from the track’s underlying random non-thermal fluctuations.

She envisions the technology as applicable beyond probing biophysical questions, in the design of 'active' technical materials.
“Imagine a microscopic biomedical device that mixes tiny samples of blood with reagents to detect disease or smart filters that separate squishy from rigid materials.”
Fakhri will join the Physics of Living Systems group, seemingly on such a bio-materials science project. MIT lab colleague Jeremy England, known for work showing that E. coli reproduction is close to thermodynamic limits of efficiency, spoke of common interest in the cytosol and diffusive processes.

“We’re interested in the non-equilibrium thermodynamics of biological organization, so that could be construed to be about evolution and the origins of life or just about how you make or design self-replicators with desired properties.”
“Increasingly there are now instruments where you can make quantitative measurements on fluorescently labeled proteins in live cells,” England explains. “The cell biologists have their language and their frame of analysis that they’re most comfortable with for describing the phenomenon, but if there are interesting phenomena that are only going to be identifiable if you do the right quantitative analysis on all these numbers that you can now measure in the cell, then it’s useful to have people who are a bit more theoretically minded or physics minded who are there, when rubber meets road, when the data is being generated and helping to influence what kind of experiments get done.”
“We’re looking, for example, at diffusion of proteins in cells. Diffusion as a qualitative phenomenon is just things spreading out over space, but as a quantitative phenomenon, you can look at things like how rapidly a protein that’s labeled over here in the cell will wander over to another region of the cell that’s a certain distance away, and if you can make measurements of that, then you can start to say things that are more specific about characteristics of the diffusion that you are observing than simply seeing it spread out. And in those quantitative measurements, you can sometimes then see differences perhaps between different cells, or different conditions for the same type of cell, that may have biological relevance but that you wouldn’t have necessarily identified without the quantitative analysis,” England says.

⌇  Fakhri et al. (2014) High-resolution mapping of intracellular fluctuations using carbon nanotubes. Science, 344(1687), 1031-5
See also:⌇  Levine and MacKintosh (2009) The mechanics and fluctuation spectrum of active gels. J Phys Chem B, 113, 3820–3830⌇  MacKintosh and Levine (2008) Nonequilibrium mechanics and dynamics of motor-activated gels. Phys Rev Lett, 100, 018104⌇  Lau et al. (2003) Microrheology, stress fluctuations, and active behavior of living cells. Phys Rev Lett, 91, 198101⇢  Related post : water’s SED failure in molecular orientational diffusion
Molecular motions inside the cell
A paper in Science this week describes the use of carbon nanotubes to pinpoint the movements of the living cell in fine detail, making for a really nice study in quantitative/mathematical biology.

Noninvasive tracking was accomplished by imaging highly stable near-infrared luminescence of single-walled carbon nanotubes targeted to kinesin-1 motor proteins in COS-7 cells. We observed a regime of active random “stirring” that constitutes an intermediate mode of transport, different from both thermal diffusion and directed motor activity. High-frequency motion was found to be thermally driven. At times greater than 100 milliseconds, nonequilibrium dynamics dominated. In addition to directed transport along microtubules, we observed strong random dynamics driven by myosins that result in enhanced nonspecific transport. We present a quantitative model connecting molecular mechanisms to mesoscopic fluctuations.

The “mesoscopic" scale is more often seen in the context of pure and applied physics (microelectronics, nanofabrication and nanotechnology), though journals such as Soft Matter present research articles giving the same ‘condensed matter’ treatment to biological systems (“Where physics meets chemistry meets biology”).
From ancient Greek μέσος it refers simply to a ‘middle’/intermediate between the molecular and macroscopic scale, where neither atomistic/quantum nor classical physics/bulk models best describe observed behaviour, and novel effects may be described — from interference effects, to quantum confinement (giving rise to band gaps) and charging effects (such as the Coulomb blockade/staircase).
Although often presented as a water-based solvent, the cytosol is more accurately described as a “highly dynamic composite material ” with mechanical properties dominated by microtubules (MTs), F-actin and intermediate filaments; all driven by metabolism-energised polymerisation of actin and tubulin and from motor proteins (specifically nucleotide triphosphate hydrolysis).
The traditional technique to observe cells in motion is fluorescence microscopy, though long-term tracking of single molecules has been hindered by fluorophores’ instabilities and the fluorescence background in cells.
Though biological networks have been termed ‘scale-free’ or ‘-invariant’, and metabolic rate for example is well known to follow a power law, the internal structure of the cell itself is far from self-similar across scales. 

At short times (microseconds to milliseconds), thermal motions should dominate. Between milliseconds and seconds, thermal diffusion might still be relevant, but there is mounting evidence, both in vitro and in vivo, that the motion of larger objects couples to myosin-driven stress fluctuations in the cytoskeleton.

»  Mizuno (2007) Nonequilibrium mechanics of active cytoskeletal networks.
»  Brangwynne (2008) Cytoplasmic diffusion: Molecular motors mix it up.

Here, temporal fluctuations, reminiscent of thermal diffusion in liquids, can arise from nonequilibrium dynamics in the viscoelastic cytoskeleton. On longer time scales, from minutes to hours, directed transport and larger-scale collective motions typically dominate. The motion of probe particles tracked inside cells has been classified as subdiffusive, diffusive, or superdiffusive. Such classifications, however, obscure the distinction between thermally driven and nonequilibrium fluctuations and are inadequate to identify intracellular material properties.

Motor proteins direct a whole host of molecular motions, kinesins and myosins being among the most heavily studied in vitro. Using fluorescence microscopy to track individual motor proteins is not only limited by instability of fluorophores, but the quality of the images taken (“signal to noise”) and efficiency of targetting probes to specific molecules.
Modern optical equipment and carefully designed fluorescent dyes have enabled experiments tracking single molecules at a time, though in living cells the authors note these experiments’ timeframes have been limited to around a second.

Their solution was to use single-walled carbon nanotubes (SWNTs), “stiff quasi–one-dimensional tubular all-carbon nanostructures with diameters of ~1 nm and persistence lengths above 10 μm” — which have the convenient property of luminescence in the near-infrared, a region ‘virtually free of autofluorescence in biological tissues’. Not only this, but the excitation time is ~100ps, such that high excitation can give ~1 ms resolution (1 ms = 109 ps).
The nanotubes were dispersed throughout the cell wrapped in short DNA oligonucleotides, with HaloTag protein fusion tags covalently attaching them specifically onto kinesin motor proteins (see Fig. 1, above).

Besides observing directed kinesin-driven transport on MTs, it is possible to directly observe fluctuations of the MT network because a moving kinesin must be bound to a MT. The MT tracks are embedded in the viscoelastic actin cytoskeleton, which in turn fluctuates as a result of stresses generated by cytoplasmic myosins.


With just 100 per cell, the group could track kinesin for up to an hour and a half, observing ~30% of them moving with some sense of direction; the rest locally constrained and moving in a random (stochastic) manner.
Some of the kinesins moved the whole length of the cell, suggesting they had cargo vesicles [along with other motor proteins] attached. Calculating mean squared displacement, MSD, of the molecules’ trajectories showed it grew over time following a power law which could be used to characterise the motion, 〈Δr2(τ)〉∝ τα(where r is distance travelled in the focal plane and τ the lag time). The exponent α shifted from ¼ to 1 between 5 ms and 2.5 s, indicating clear scale variance to the motion.
After this, the group acquired the nanotubes’ fluorescent signal at a rate of four frames per second ‒ using this 250 ms window to observe an intermediate between the thermal diffusion seen on the short timescales and directed motor activity.
With a well-designed control or two, they showed that the transverse motion of the nanotube-marked microtubules was not due to kinesin motors, but reflecting intrinsic dynamics of the cytoskeleton.

The way the relatively rigid MTs report these dynamics depends on two restoring forces: the elastic force of bent MTs and the force exerted by the strained cytoskeletal matrix in which the MTs are embedded. Because it is hard to bend an elastic rod on short length scales, the surrounding matrix yields to the MT when it is deformed on short length scales. By contrast, the MT yields to matrix forces for deflections of wavelength larger than ~1 μm. The shorter-wavelength MT deflections relax faster than our 5-ms frame rate. Therefore, we assume that the transverse MT motion we observe reflects the (active or passive) strain fluctuations of the surrounding matrix.
The MSD power-law exponent α generally reflects the randomness of motion. More precisely, in any medium, the MSD of an embedded probe particle is governed both by the material properties of the medium and the temporal characteristics of the forces driving the particle. For thermally driven Brownian motion in simple liquids, the MSD exponent α = 1. For thermal motion in viscoelastic media, which exhibit time- and frequency-dependent viscosity and elasticity, α < 1 strictly holds. For viscoelastic materials, the stiffness G(ω) typically increases with a power of frequency ω: G(ω) ∝ ωβ. This is observed in polymer solutions, where the viscoelastic exponent β ≈ 0.5 to 0.8, as well as in cells, where β ≈ 0.1 to 0.2 on time scales on the order of seconds. This value of the exponent is close to what is expected for purely elastic materials, where β = 0. 
The nearly elastic behavior of cells can be understood as a consequence of strong cross-linking in the cytoskeleton.
 Knowing the driving forces, it is possible to construct a relation between MSD exponent α and viscoelastic exponent β. For thermal driving forces, the MSD exponent α = β. Thermal fluctuations can therefore never appear as “superdiffusive” motion with α > 1. Nonthermal driving, by contrast, can result in superdiffusive motion. Theory provides a specific prediction for motion in nearly elastic solids driven by random stress fluctuations with long correlation times and sudden transitions: α = 1 + 2β. This prediction is expected to apply for cytoskeletal stress fluctuations caused by randomly distributed cytoplasmic myosin minifilaments. Myosin locally contracts the actin network with an attachment time of several seconds, followed by sudden release. Some hints of this predicted scaling have been reported for cells and reconstituted acto-myosin model systems. When β = 0 (i.e., in the elastic limit), the resulting MSDs can look deceptively like Brownian motion in a simple liquid, although the physical reason is entirely different. For observation times τ longer than the correlation time of the driving forces, the MSD is predicted to level off, as we observed. In our experiments, the stress correlation time should correspond to typical cytoplasmic myosin motor engagement times, which are indeed reported to be ~10 s in cells.

Still attached to microtubules, the kinesin molecules exhibit vigorous random (Brownian-like) motion as they are buffeted by myosins as described ‒ likely thrusting MTs into the path of other cellular particles. Tubulin forms strong tubular filaments embedded in a more flexible actin network. Nonmuscle myosin II exerts mechanical stress on it, which is released ‘suddenly’ as random stirring of the whole filament network, including the microtubules.

We observed a transition between thermal dynamics in the dominantly elastic cytoskeleton at short times to strongly nonequilibrium power-law dynamics, likely driven by myosin activity, at intermediate times. When the time exceeded the correlation time of the random stress generators, the intermediate regime was followed by a saturation to a maximum MSD, nearly constant over time. Note that in this regime, the MSD amplitude corresponds to a root mean square displacement of ~500 nm, which is larger than the estimated mesh size of the actin network, and thus larger than the expected spacing of obstacles in the crowded cytoplasm.

The authors lastly used myosin inhibitor blebbistatin to block myosin from the actin network, confirming their hypothesis with a dose-dependent reduction in what they call the amplitude of active stirring, and exponent α, “establishing nonmuscle myosin II as the dominant driving factor for random cytoskeletal stirring”.

We can explain the regimes we observe by a quantitative model of cytoskeletal fluctuations and directed motor motion that describes the transition from thermal motion to nonequilibrium stirring dynamics driven by myosin, as well as the transition from stirring dynamics to directed transport driven by kinesin. Our observations were made possible by the use of SWNT labels for broadband molecular tracking in cells. Many questions concerning motor transport in cells will now be addressable using this approach. We have focused here on the stirring dynamics, which constitute an important mode of active intracellular transport between the limits of random thermal diffusion and directed transport, accelerating nonspecific transport through the nanoporous cytoskeleton.

Lead author Nikta Fakhri will soon leave the Göttingen Institute for Biophysics to join the faculty at MIT as assistant professor of physics. Fakhri gave a talk in Massachussets last year on the topic, to the Chemical Engineering department in which some of the details of this paper made their debut:

The discovery of fullerenes provided exciting insights into how highly symmetrical structures of pure carbon can have remarkable physical properties. Single-walled carbon nanotubes (SWNTs) are the vanguard of such architectures. The organization of the hexagonal honeycomb carbon lattice into high-aspect-ratio cylinders with a variety of helical symmetries creates very unusual macromolecular structures representing an emerging research area in condensed matter physics and materials science: traditionally hard materials appearing in new soft matter applications and environments.
… the dynamics of SWNTs in liquids are essentially polymer-like. By exploiting the intrinsic near-infrared fluorescence of semiconducting SWNTs, we have imaged the Brownian motion of individual nanotubes in water and have measured directly the bending stiffness of SWNTs. The semiflexible chain model represents accurately the configurational dynamics of SWNTs suspended in water. Interestingly, the persistence length of SWNTs is comparable to that of biopolymers. This finding paves the way for using SWNTs as a model system for semiflexible polymers to answer long-standing fundamental questions in polymer physics.
… the confined dynamics of stiff macromolecules in crowded environments [are] a common feature of polymer composites and the cell cytoskeleton. In fixed porous networks, we find that even a small bending flexibility strongly enhances SWNTs’ motion. This ends a 30-year-old debate in polymer physics: the rotational diffusion constant is proportional to the filament bending compliance and counter-intuitively, independent of the network porosity. The dynamics of SWNTs in equilibrium and non-equilibrium biopolymer networks is more complex.
At long times, SWNTs reptate in the networks. At short times SWNTs can sample the spectrum of local stresses in equilibrium networks. In the non-equilibrium networks we observe strong local shape fluctuations driven by force generating molecular motors. I will discuss a newly developed microrheology technique in which we use nanotubes as “stealth probes” to measure viscoelastic properties of the host media. Finally, I will introduce a new single-molecule technique based on ultra-stable near-infrared fluorescence of short SWNTs, to study intracellular transport dynamics in living cells and in whole organisms. The combination of long-time stability and high signal-to-noise ratio enables the accurate long-term tracking of single motor proteins tagged with SWNTs traversing the entire cell. Remarkably, we can distinguish the motor protein’s motion along its microtubule track from the track’s underlying random non-thermal fluctuations.

She envisions the technology as applicable beyond probing biophysical questions, in the design of 'active' technical materials.
“Imagine a microscopic biomedical device that mixes tiny samples of blood with reagents to detect disease or smart filters that separate squishy from rigid materials.”
Fakhri will join the Physics of Living Systems group, seemingly on such a bio-materials science project. MIT lab colleague Jeremy England, known for work showing that E. coli reproduction is close to thermodynamic limits of efficiency, spoke of common interest in the cytosol and diffusive processes.

“We’re interested in the non-equilibrium thermodynamics of biological organization, so that could be construed to be about evolution and the origins of life or just about how you make or design self-replicators with desired properties.”
“Increasingly there are now instruments where you can make quantitative measurements on fluorescently labeled proteins in live cells,” England explains. “The cell biologists have their language and their frame of analysis that they’re most comfortable with for describing the phenomenon, but if there are interesting phenomena that are only going to be identifiable if you do the right quantitative analysis on all these numbers that you can now measure in the cell, then it’s useful to have people who are a bit more theoretically minded or physics minded who are there, when rubber meets road, when the data is being generated and helping to influence what kind of experiments get done.”
“We’re looking, for example, at diffusion of proteins in cells. Diffusion as a qualitative phenomenon is just things spreading out over space, but as a quantitative phenomenon, you can look at things like how rapidly a protein that’s labeled over here in the cell will wander over to another region of the cell that’s a certain distance away, and if you can make measurements of that, then you can start to say things that are more specific about characteristics of the diffusion that you are observing than simply seeing it spread out. And in those quantitative measurements, you can sometimes then see differences perhaps between different cells, or different conditions for the same type of cell, that may have biological relevance but that you wouldn’t have necessarily identified without the quantitative analysis,” England says.

⌇  Fakhri et al. (2014) High-resolution mapping of intracellular fluctuations using carbon nanotubes. Science, 344(1687), 1031-5
See also:⌇  Levine and MacKintosh (2009) The mechanics and fluctuation spectrum of active gels. J Phys Chem B, 113, 3820–3830⌇  MacKintosh and Levine (2008) Nonequilibrium mechanics and dynamics of motor-activated gels. Phys Rev Lett, 100, 018104⌇  Lau et al. (2003) Microrheology, stress fluctuations, and active behavior of living cells. Phys Rev Lett, 91, 198101⇢  Related post : water’s SED failure in molecular orientational diffusion

Molecular motions inside the cell

A paper in Science this week describes the use of carbon nanotubes to pinpoint the movements of the living cell in fine detail, making for a really nice study in quantitative/mathematical biology.

Noninvasive tracking was accomplished by imaging highly stable near-infrared luminescence of single-walled carbon nanotubes targeted to kinesin-1 motor proteins in COS-7 cells. We observed a regime of active random “stirring” that constitutes an intermediate mode of transport, different from both thermal diffusion and directed motor activity. High-frequency motion was found to be thermally driven. At times greater than 100 milliseconds, nonequilibrium dynamics dominated. In addition to directed transport along microtubules, we observed strong random dynamics driven by myosins that result in enhanced nonspecific transport. We present a quantitative model connecting molecular mechanisms to mesoscopic fluctuations.

The “mesoscopic" scale is more often seen in the context of pure and applied physics (microelectronics, nanofabrication and nanotechnology), though journals such as Soft Matter present research articles giving the same ‘condensed matter’ treatment to biological systems (“Where physics meets chemistry meets biology”).

From ancient Greek μέσος it refers simply to a ‘middle’/intermediate between the molecular and macroscopic scale, where neither atomistic/quantum nor classical physics/bulk models best describe observed behaviour, and novel effects may be described — from interference effects, to quantum confinement (giving rise to band gaps) and charging effects (such as the Coulomb blockade/staircase).

Although often presented as a water-based solvent, the cytosol is more accurately described as a “highly dynamic composite material ” with mechanical properties dominated by microtubules (MTs), F-actin and intermediate filaments; all driven by metabolism-energised polymerisation of actin and tubulin and from motor proteins (specifically nucleotide triphosphate hydrolysis).

The traditional technique to observe cells in motion is fluorescence microscopy, though long-term tracking of single molecules has been hindered by fluorophores’ instabilities and the fluorescence background in cells.

Though biological networks have been termed ‘scale-free’ or ‘-invariant’, and metabolic rate for example is well known to follow a power law, the internal structure of the cell itself is far from self-similar across scales. 

At short times (microseconds to milliseconds), thermal motions should dominate. Between milliseconds and seconds, thermal diffusion might still be relevant, but there is mounting evidence, both in vitro and in vivo, that the motion of larger objects couples to myosin-driven stress fluctuations in the cytoskeleton.

»  Mizuno (2007) Nonequilibrium mechanics of active cytoskeletal networks.

»  Brangwynne (2008) Cytoplasmic diffusion: Molecular motors mix it up.

Here, temporal fluctuations, reminiscent of thermal diffusion in liquids, can arise from nonequilibrium dynamics in the viscoelastic cytoskeleton. On longer time scales, from minutes to hours, directed transport and larger-scale collective motions typically dominate. The motion of probe particles tracked inside cells has been classified as subdiffusive, diffusive, or superdiffusive. Such classifications, however, obscure the distinction between thermally driven and nonequilibrium fluctuations and are inadequate to identify intracellular material properties.

Motor proteins direct a whole host of molecular motions, kinesins and myosins being among the most heavily studied in vitro. Using fluorescence microscopy to track individual motor proteins is not only limited by instability of fluorophores, but the quality of the images taken (“signal to noise”) and efficiency of targetting probes to specific molecules.

Modern optical equipment and carefully designed fluorescent dyes have enabled experiments tracking single molecules at a time, though in living cells the authors note these experiments’ timeframes have been limited to around a second.

Their solution was to use single-walled carbon nanotubes (SWNTs), “stiff quasi–one-dimensional tubular all-carbon nanostructures with diameters of ~1 nm and persistence lengths above 10 μm” — which have the convenient property of luminescence in the near-infrared, a region ‘virtually free of autofluorescence in biological tissues’. Not only this, but the excitation time is ~100ps, such that high excitation can give ~1 ms resolution (1 ms = 109 ps).

The nanotubes were dispersed throughout the cell wrapped in short DNA oligonucleotides, with HaloTag protein fusion tags covalently attaching them specifically onto kinesin motor proteins (see Fig. 1, above).

Besides observing directed kinesin-driven transport on MTs, it is possible to directly observe fluctuations of the MT network because a moving kinesin must be bound to a MT. The MT tracks are embedded in the viscoelastic actin cytoskeleton, which in turn fluctuates as a result of stresses generated by cytoplasmic myosins.

image

With just 100 per cell, the group could track kinesin for up to an hour and a half, observing ~30% of them moving with some sense of direction; the rest locally constrained and moving in a random (stochastic) manner.

Some of the kinesins moved the whole length of the cell, suggesting they had cargo vesicles [along with other motor proteins] attached. Calculating mean squared displacement, MSD, of the molecules’ trajectories showed it grew over time following a power law which could be used to characterise the motion, 〈Δr2(τ)〉∝ τα(where r is distance travelled in the focal plane and τ the lag time). The exponent α shifted from ¼ to 1 between 5 ms and 2.5 s, indicating clear scale variance to the motion.

After this, the group acquired the nanotubes’ fluorescent signal at a rate of four frames per second ‒ using this 250 ms window to observe an intermediate between the thermal diffusion seen on the short timescales and directed motor activity.

With a well-designed control or two, they showed that the transverse motion of the nanotube-marked microtubules was not due to kinesin motors, but reflecting intrinsic dynamics of the cytoskeleton.

The way the relatively rigid MTs report these dynamics depends on two restoring forces: the elastic force of bent MTs and the force exerted by the strained cytoskeletal matrix in which the MTs are embedded. Because it is hard to bend an elastic rod on short length scales, the surrounding matrix yields to the MT when it is deformed on short length scales. By contrast, the MT yields to matrix forces for deflections of wavelength larger than ~1 μm. The shorter-wavelength MT deflections relax faster than our 5-ms frame rate. Therefore, we assume that the transverse MT motion we observe reflects the (active or passive) strain fluctuations of the surrounding matrix.

The MSD power-law exponent α generally reflects the randomness of motion. More precisely, in any medium, the MSD of an embedded probe particle is governed both by the material properties of the medium and the temporal characteristics of the forces driving the particle. For thermally driven Brownian motion in simple liquids, the MSD exponent α = 1. For thermal motion in viscoelastic media, which exhibit time- and frequency-dependent viscosity and elasticity, α < 1 strictly holds. For viscoelastic materials, the stiffness G(ω) typically increases with a power of frequency ω: G(ω) ∝ ωβ. This is observed in polymer solutions, where the viscoelastic exponent β ≈ 0.5 to 0.8, as well as in cells, where β ≈ 0.1 to 0.2 on time scales on the order of seconds. This value of the exponent is close to what is expected for purely elastic materials, where β = 0.

The nearly elastic behavior of cells can be understood as a consequence of strong cross-linking in the cytoskeleton.

Knowing the driving forces, it is possible to construct a relation between MSD exponent α and viscoelastic exponent β. For thermal driving forces, the MSD exponent α = β. Thermal fluctuations can therefore never appear as “superdiffusive” motion with α > 1. Nonthermal driving, by contrast, can result in superdiffusive motion. Theory provides a specific prediction for motion in nearly elastic solids driven by random stress fluctuations with long correlation times and sudden transitions: α = 1 + 2β. This prediction is expected to apply for cytoskeletal stress fluctuations caused by randomly distributed cytoplasmic myosin minifilaments. Myosin locally contracts the actin network with an attachment time of several seconds, followed by sudden release. Some hints of this predicted scaling have been reported for cells and reconstituted acto-myosin model systems. When β = 0 (i.e., in the elastic limit), the resulting MSDs can look deceptively like Brownian motion in a simple liquid, although the physical reason is entirely different. For observation times τ longer than the correlation time of the driving forces, the MSD is predicted to level off, as we observed. In our experiments, the stress correlation time should correspond to typical cytoplasmic myosin motor engagement times, which are indeed reported to be ~10 s in cells.

Still attached to microtubules, the kinesin molecules exhibit vigorous random (Brownian-like) motion as they are buffeted by myosins as described ‒ likely thrusting MTs into the path of other cellular particles. Tubulin forms strong tubular filaments embedded in a more flexible actin network. Nonmuscle myosin II exerts mechanical stress on it, which is released ‘suddenly’ as random stirring of the whole filament network, including the microtubules.

We observed a transition between thermal dynamics in the dominantly elastic cytoskeleton at short times to strongly nonequilibrium power-law dynamics, likely driven by myosin activity, at intermediate times. When the time exceeded the correlation time of the random stress generators, the intermediate regime was followed by a saturation to a maximum MSD, nearly constant over time. Note that in this regime, the MSD amplitude corresponds to a root mean square displacement of ~500 nm, which is larger than the estimated mesh size of the actin network, and thus larger than the expected spacing of obstacles in the crowded cytoplasm.

The authors lastly used myosin inhibitor blebbistatin to block myosin from the actin network, confirming their hypothesis with a dose-dependent reduction in what they call the amplitude of active stirring, and exponent α, “establishing nonmuscle myosin II as the dominant driving factor for random cytoskeletal stirring”.

We can explain the regimes we observe by a quantitative model of cytoskeletal fluctuations and directed motor motion that describes the transition from thermal motion to nonequilibrium stirring dynamics driven by myosin, as well as the transition from stirring dynamics to directed transport driven by kinesin. Our observations were made possible by the use of SWNT labels for broadband molecular tracking in cells. Many questions concerning motor transport in cells will now be addressable using this approach. We have focused here on the stirring dynamics, which constitute an important mode of active intracellular transport between the limits of random thermal diffusion and directed transport, accelerating nonspecific transport through the nanoporous cytoskeleton.

Lead author Nikta Fakhri will soon leave the Göttingen Institute for Biophysics to join the faculty at MIT as assistant professor of physics. Fakhri gave a talk in Massachussets last year on the topic, to the Chemical Engineering department in which some of the details of this paper made their debut:

The discovery of fullerenes provided exciting insights into how highly symmetrical structures of pure carbon can have remarkable physical properties. Single-walled carbon nanotubes (SWNTs) are the vanguard of such architectures. The organization of the hexagonal honeycomb carbon lattice into high-aspect-ratio cylinders with a variety of helical symmetries creates very unusual macromolecular structures representing an emerging research area in condensed matter physics and materials science: traditionally hard materials appearing in new soft matter applications and environments.

… the dynamics of SWNTs in liquids are essentially polymer-like. By exploiting the intrinsic near-infrared fluorescence of semiconducting SWNTs, we have imaged the Brownian motion of individual nanotubes in water and have measured directly the bending stiffness of SWNTs. The semiflexible chain model represents accurately the configurational dynamics of SWNTs suspended in water. Interestingly, the persistence length of SWNTs is comparable to that of biopolymers. This finding paves the way for using SWNTs as a model system for semiflexible polymers to answer long-standing fundamental questions in polymer physics.

… the confined dynamics of stiff macromolecules in crowded environments [are] a common feature of polymer composites and the cell cytoskeleton. In fixed porous networks, we find that even a small bending flexibility strongly enhances SWNTs’ motion. This ends a 30-year-old debate in polymer physics: the rotational diffusion constant is proportional to the filament bending compliance and counter-intuitively, independent of the network porosity. The dynamics of SWNTs in equilibrium and non-equilibrium biopolymer networks is more complex.

At long times, SWNTs reptate in the networks. At short times SWNTs can sample the spectrum of local stresses in equilibrium networks. In the non-equilibrium networks we observe strong local shape fluctuations driven by force generating molecular motors. I will discuss a newly developed microrheology technique in which we use nanotubes as “stealth probes” to measure viscoelastic properties of the host media. Finally, I will introduce a new single-molecule technique based on ultra-stable near-infrared fluorescence of short SWNTs, to study intracellular transport dynamics in living cells and in whole organisms. The combination of long-time stability and high signal-to-noise ratio enables the accurate long-term tracking of single motor proteins tagged with SWNTs traversing the entire cell. Remarkably, we can distinguish the motor protein’s motion along its microtubule track from the track’s underlying random non-thermal fluctuations.

She envisions the technology as applicable beyond probing biophysical questions, in the design of 'active' technical materials.

“Imagine a microscopic biomedical device that mixes tiny samples of blood with reagents to detect disease or smart filters that separate squishy from rigid materials.”

Fakhri will join the Physics of Living Systems group, seemingly on such a bio-materials science project. MIT lab colleague Jeremy England, known for work showing that E. coli reproduction is close to thermodynamic limits of efficiency, spoke of common interest in the cytosol and diffusive processes.

“We’re interested in the non-equilibrium thermodynamics of biological organization, so that could be construed to be about evolution and the origins of life or just about how you make or design self-replicators with desired properties.”

“Increasingly there are now instruments where you can make quantitative measurements on fluorescently labeled proteins in live cells,” England explains. “The cell biologists have their language and their frame of analysis that they’re most comfortable with for describing the phenomenon, but if there are interesting phenomena that are only going to be identifiable if you do the right quantitative analysis on all these numbers that you can now measure in the cell, then it’s useful to have people who are a bit more theoretically minded or physics minded who are there, when rubber meets road, when the data is being generated and helping to influence what kind of experiments get done.”

“We’re looking, for example, at diffusion of proteins in cells. Diffusion as a qualitative phenomenon is just things spreading out over space, but as a quantitative phenomenon, you can look at things like how rapidly a protein that’s labeled over here in the cell will wander over to another region of the cell that’s a certain distance away, and if you can make measurements of that, then you can start to say things that are more specific about characteristics of the diffusion that you are observing than simply seeing it spread out. And in those quantitative measurements, you can sometimes then see differences perhaps between different cells, or different conditions for the same type of cell, that may have biological relevance but that you wouldn’t have necessarily identified without the quantitative analysis,” England says.

⌇  Fakhri et al. (2014) High-resolution mapping of intracellular fluctuations using carbon nanotubesScience344(1687), 1031-5

See also:
⌇  Levine and MacKintosh (2009) The mechanics and fluctuation spectrum of active gels. J Phys Chem B, 113, 3820–3830
⌇  MacKintosh and Levine (2008) Nonequilibrium mechanics and dynamics of motor-activated gels. Phys Rev Lett, 100, 018104
⌇  Lau et al. (2003) Microrheology, stress fluctuations, and active behavior of living cells. Phys Rev Lett91, 198101

⇢  Related post : water’s SED failure in molecular orientational diffusion

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I know that it is a hopeless undertaking to debate about fundamental value judgments. For instance if someone approves, as a goal, the extirpation of the human race from the earth, one cannot refute such a viewpoint on rational grounds. But if there is agreement on certain goals and values, one can argue rationally about the means by which these objectives may be attained. Let us, then, indicate two goals which may well be agreed upon by nearly all who read these lines.

  1. Those instrumental goods which should serve to maintain the life and health of all human beings should be produced by the least possible labor of all.

  2. The satisfaction of physical needs is indeed the indispensable precondition of a satisfactory existence, but in itself it is not enough. In order to be content men must also have the possibility of developing their intellectual and artistic powers to whatever extent accord with their personal characteristics and abilities.

The first of these two goals requires the promotion of all knowledge relating to the laws of nature and the laws of social processes, that is, the promotion of all scientific endeavor. For scientific endeavor is a natural whole the parts of which mutually support one another in a way which, to be sure, no one can anticipate. However, the progress of science presupposes the possibility of unrestricted communication of all results and judgments—freedom of expression and instruction in all realms of intellectual endeavor. By freedom I understand social conditions of such a kind that the expression of opinions and assertions about general and particular matters of knowledge will not involve dangers or serious disadvantages for him who expresses them. This freedom of communication is indispensable for the development and extension of scientific knowledge, a consideration of much practical import. In the first instance it must be guaranteed by law. But laws alone cannot secure freedom of expression; in order that every man may present his views without penalty there must be a spirit of tolerance in the entire population. Such an ideal of external liberty can never be fully attained but must be sought unremittingly if scientific thought, and philosophical and creative thinking in general, are to be advanced as far as possible.

If the second goal, that is, the possibility of the spiritual development of all individuals, is to be secured, a second kind of outward freedom is necessary. Man should not have to work for the achievement of the necessities of life to such an extent that he has neither time nor strength for personal activities. Without this second kind of outward liberty, freedom of expression is useless for him. Advances in technology would provide the possibility of this kind of freedom if the problem of a reasonable division of labor were solved.

The development of science and of the creative activities of the spirit in general requires still another kind of freedom, which may be characterized as inward freedom. It is this freedom of the spirit which consists in the independence of thought from the restrictions of authoritarian and social prejudices as well as from unphilosophical routinizing and habit in general. This inward freedom is an infrequent gift of nature and a worthy objective for the individual. Yet the community can do much to further this achievement, too, at least by not interfering with its development. Thus schools may interfere with the development of inward freedom through authoritarian influences and through imposing on young people excessive spiritual burdens; on the other hand schools may favor such freedom by encouraging independent thought. Only if outward and inner freedom are constantly and consciously pursued is there a possibility of spiritual development and perfection and thus of improving man’s outward and inner life.

“On Freedom” by Albert Einstein, in the anthology of his essays Out of My Later Years.

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Overlay journals and supplement review

An overlay journal (or repository journal) is a specific type of open access journal (generally online). While not producing content itself, it selects from texts that are freely available ‒ deriving content from preprint servers such as arXiv, or from commercial publishers using self-archived, pre-, or post-prints.

Editors locate and evaluate suitable material, anywhere from the decision of a single editor up to a full peer review process. Brown (2010) writes that

An overlay journal performs all the activities of a scholarly journal and relies on structural links with one or more archives or repositories to perform its activities.

while Peter Suber (2003) defines one as “an open access journal that takes submissions from the preprints deposited at an archive… and subjects them to peer-review”. Referring to them as repository journals, he also wrote that they

use the institutional repository as the journal infrastructure. Submissions are deposited in the journal as preprints, and accepted articles are redeposited as postprints, labelled to show that they have been peer-reviewed.

In a footnote, Harley and Acord (2011) detail how this new phenomenon is “still fairly speculative at present

An overlay journal would mine self-archived “raw” author manuscripts from repositories and carry out certain publishing functions like peer-review management, editing, and perhaps branding (Swan 2010). The actual published content would continue to reside in the repository, perhaps with an updated postprint incorporating any revisions and updated metadata reflecting the journal/society brand that carried out the peer review. The overlay journal would then link to the repository content via a traditional table of contents.

…minimalist journals that provide peer review but not a publishing platform.

The emphasis therefore is on creating the canon of ‘prized’ literature, seemingly in response to the insidious practice of valuation according to use (citation) and location (quantified as ‘impact’ in the Impact Factor). Whether any editing would take place is an interesting question.

In a formal remarks section of this discussion paper, librarian (and Dean of Libraries, Professor of Public Policy, Economics, and Information, Former Provost and Executive Vice President for Academic Affairs!) Paul Courant at the University of Michigan adds his thoughts.

Why change the peer-review publishing system if mandates and Green OA can solve the problem of open access?

I would be surprised if mandates for open access would solve the problem; that would imply significant change in the relationship between peer review and publication. I think what is imagined by this question is that the institutional repositories to which people are making deposits serve as a bin from which to create overlay journals.

I do wonder whether having heavily populated institutional repositories, which were open to the world and for non-commercial republishing of various kinds, would solve a lot of the problem. I think that an as likely route would be if the federal government mandated an open access version of all scholarship produced with federal funds within six months or a year of publication, and that that version is allowed to be read and recombined in various ways. That would certainly have a salutary effect on the way the commercial publishing industry works. Additionally, universities should reserve a slice of copyright so that they can always make that happen.

Lior Pachter, a computational biologist who notably published the Cufflinks RNA-seq transcript assembly software, has spoken of science’s lapsed obligation to its ‘supplementary’ data (those research data that are moved out of the main article, and as such often neglected during peer review).

These limitations arise from the traditional scholarly publishing system, but with literature now both read and submitted electronically it seems counterproductive to still be doing so.

To replace this facet of the research process, Lior writes his own blog, and draws regular comment from others in the field. This type of activitity is usually pitched as post-publication peer review, though I’m yet to hear of any other avenues through which supplementary materials could be subjected to scrutiny and held to adequate account.

It seems quite important to me for previous research to be discussed and posts of worth to be brought to the attention of others in a way that steps outside of the publication-PR cycle. The push for (and increasing presence of) open access in academia certainly makes the ability to change the canon of ‘top literature’ more feasible — something both overlay journals and post-publication peer review aim to achieve.

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It is difficult to obtain conclusive proof of absence of transcription because transcription may only occur under particular environmental conditions that do not match those assayed in the laboratory.

Wilson & Masel (2011), in a paper also detailing the potential for “a simple product of chance or phylogenetic confounding” to undermine conclusions drawn from lab assays.


Professor of Biology and Biochemistry at the University of Houston Dan Graur, who writes as judgestarling, pointed out this neat review from 2011, when prompted for an example of transcribed and translated “junk” DNA.

Another paper has been published by Manolis Kellis et al, members of the ENCODE project which set out to define, describe and quantify function in the genome (the dispute over which I covered here last year).

The developments in this story aside, this paper details how putatively noncoding transcripts show extensive association with ribosomes. Whole genes arising de novo from non-coding DNA amounts to a sort of virgin birth in evolutionary terms — at odds with gradual change and adaptation.

There have been recent surprising reports that whole genes can evolve de novo from noncoding sequences. This would be extraordinary if the noncoding sequences were random with respect to amino acid identity. However, if the noncoding sequences were previously translated at low rates, with the most strongly deleterious cryptic polypeptides purged by selection, then de novo gene origination would be more plausible.

I’ll write more on this soon, in the mean time the paper is open access at the link below, and for more on de novo gene formation see my previous post and Carl Zimmer’s recent piece in the New York times in which he speaks to Christian Schlötterer, the author of the eLife paper I mentioned midway through.

 Wilson, B. & Masel, J. (2011) Putatively noncoding transcripts show extensive association with ribosomes. Genome Biology & Evolution, 3, 1245-1252

Masel had previously written a more mathematical paper on fitness, and a “local solution [that] facilitates the genetic assimilation of cryptic genetic variation”:
  Rajon and Masel (2011) Evolution of molecular error rates and the consequences for evolvability. PNAS, 108(3) 1082‒1087

⇢  The Masel group’s web page has further details on this and their other lines of research, including the connection between de novo gene birth and the need for preventing aggregation as a constraint on sequence evolution therein

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E-cigarettes: what&#8217;s the harm&#160;?
Tobacco smoking is now a globally accepted public health issue, and recently e-cigarettes have emerged as a socially acceptable form of an increasingly marginalised habit. They occupy a somewhat grey area, with those in medicine unsure whether to slate them if they might be a tool in getting hardcore smokers to quit the obviously more contaminated and harmful paper counterparts.
The BBC reports that e-cigarette users have tripled since 2010 (based on health charity Ash's estimates). The group say that use amongst non-smokers remains small (~1%).
NHS Choices gives a good impartial run-down of the organisation&#8217;s report, which highlights that roughly  ⅓ of e-cig users are ex- and ⅔ are current smokers. The NHS stresses that although this survey may capture the negligible rate of use among non-smokers now, it&#8217;s impossible to predict how use of such a new technology may develop.
Associate editor of the British Medical Journal Douglas Kamerow observed earlier this month that:

one can envisage three possible scenarios in which e-cigarettes are used: one good, two bad. Potentially, they could be useful to help tobacco smokers quit, replacing cigarettes and slowly dialing down nicotine levels, much as other nicotine delivery devices (gum, patches, inhalers, and so on) do. That would be a good thing, although the research that is starting to accumulate about this therapeutic use of e-cigarettes is so far not promising.
Secondly, e-cigarettes could be used to maintain a tobacco habit by allowing smokers nicotine intake in tobacco free areas, such as the workplace or home, because the vapor emitted from e-cigarettes is odorless and presumed to be not toxic. This is not so good for public health, as one of the most important incentives for quitting smoking has been the dramatic increase in places that prohibit it.
Thirdly, and most distressing, e-cigarettes could be used to initiate nicotine use and perhaps lead to tobacco smoking. With their many flavors and nicotine levels, they are easy to use and appealing to vapers of all ages, including adolescents. Friends report that an e-cigarette is the new status item in middle schools here, where they are easily concealed and used in bathrooms and outdoor breaks. Will this lead to an end or reversal of the long term downward trend in tobacco use? Time will tell. But the latest trend in e-cigarettes is perhaps more frightening still. Most early e-cigarettes were disposable appliances designed to look and feel like cigarettes; they even had a little LED light at the end that lit up when you inhaled. You used them up and then threw them away. What we’re seeing now, however, is larger vaporizing systems. Their batteries can be recharged with the USB port of your computer and the vaporizer refilled with nicotine liquid (called “juice”) in your choice of flavor at your preferred nicotine concentration.
The problem is thatnicotine is a poison, and the e-liquid is easily absorbed in several ways: inhalation, certainly, but also through ingestion and even skin contact. Add in the fact that the “juices” come in cute little bottles of colored, great smelling liquids and you have the makings of a terrific attraction for toddlers and young children. Unsurprisingly, US poison control centers are reporting threefold increases in calls about e-liquid poisonings. A slug of 36&#160;mg/mL of e-liquid can make a child very sick. No deaths have been reported yet, but it is only a matter of time. Higher concentrations of e-liquid, available wholesale in gallon jugs on the internet for home brewing, are deadlier yet. And childrenaren’t theonlyvictims. Emergencyrooms are reporting adults showing up with cardiac symptoms after spilling e-liquid on their skin.

As a doctor, it must be hard to live through a generation that&#8217;s fought off the lawsuits and marketing ploys of Big Tobacco and its shifty lobbies even to just achieve scientific consensus on the harm smoking does, and then to see a 21st century version appear in its wake.
The New York Times provides further coverage of casualties in a society now keeping a toxic stimulant liquid within fragile containers.

Last month, a 2-year-old girl in Oklahoma City drank a small bottle of a parent’s nicotine liquid, started vomiting and was rushed to an emergency room.
That case and age group is considered typical. Of the 74 e-cigarette and nicotine poisoning cases called into Minnesota poison control in 2013, 29 involved children age 2 and under. In Oklahoma, all but two of the 25 cases in the first two months of this year involved children age 4 and under.
“This is one of the most potent naturally occurring toxins we have,” Mr. Cantrell said of nicotine. But e-liquids are now available almost everywhere. “It is sold all over the place. It is ubiquitous in society.”
The surge in poisonings reflects not only the growth of e-cigarettes but also a shift in technology. Initially, many e-cigarettes were disposable devices that looked like conventional cigarettes. Increasingly, however, they are larger, reusable gadgets that can be refilled with liquid, generally a combination of nicotine, flavorings and solvents. In Kentucky, where about 40 percent of cases involved adults, one woman was admitted to the hospital with cardiac problems after her e-cigarette broke in her bed, spilling the e-liquid, which was then absorbed through her skin.
The problems with adults, like those with children, owe to carelessness and lack of understanding of the risks. In the cases of exposure in children, “a lot of parents didn’t realize it was toxic until the kid started vomiting,” said Ashley Webb, director of the Kentucky Regional Poison Control Center at Kosair Children’s Hospital.


Children are often brought up as “passive” sufferers from cigarettes, but acute toxicity frames it quite starkly. Nicotine clearly has its dangers, but to put that aside for a moment, a study of the contaminants in e-cigarettes found nothing above workplace standards of toxicity.
Smoking has largely moved out of the media spotlight but its spectre looms large as the leading cause of cancer deaths in the world today
It&#8217;s been sidelined from film, television and advertising, and in many countries banned from indoor spaces. Responsible for 90% of all lung cancers, the most common of the world&#8217;s cancer, there remain too few indicators of who is at most risk (for lung cancer or COPD), and it&#8217;s still hazy as to why the risk lingers for individuals who stubbed out years ago.
Preclinical research using human bronchial cells containing mutations found in smokers at risk of lung cancer was presented recently at the American Association for Cancer Research annual meeting.
There has been very little experimental research into the subject at all but this posting describes the effect of e-cigarette exposure on airway epithelial cell gene expression and transformation, in the spirit of earlier papers which started to unpick the mutagenesis we&#8217;re now all so aware of.
The finding suggests that the e-cig is not as benign as presented.

The toxicity and potential carcinogenicity of ECIGs have not previously been evaluated. In this study, we assess the impact of ECIG exposure on the carcinogenic potential of immortalized human bronchial epithelial cells on a background of silenced p53 and activated KRAS (H3mut-P53/KRAS). This model is utilized because p53 and KRAS mutations are often observed in the airway of current and former smokers at risk for lung cancer.

For a guided tour of tumour suppressor p53&#8217;s mechanisms of inactivation, see last week&#8217;s post. KRAS is a similar gene, though works inversely by promoting hyperproliferation upon activation — what&#8217;s known as a proto-oncogene.

The epithelial cells were exposed to both a low and high concentration of nicotine in the ECIG vapor- or tobacco cigarette (TCIG) smoke-conditioned media. The lower nicotine concentration was selected to mimic the average plasma nicotine levels in electronic nicotine delivery system (ENDS) users and did not demonstrate toxic or anti-proliferative effects on the cells. The higher concentration was chosen to represent the anticipated nicotine levels to which the epithelial cells of smokers are actually exposed. In anchorage independent growth assays, the in vitro correlate of malignant transformation, we found enhanced colony growth in the H3mut-P53/KRAS cells following a 10-day treatment with the high nicotine ECIG- and TCIG-conditioned media compared to the untreated and low nicotine treatment groups.
We next assessed the effect of ECIG and TCIG exposure on cell invasion using a three-dimensional air-liquid interface (ALI) model. At baseline, H3mut-P53/KRAS cells exhibit invasive behavior in the ALI model, due to the downstream effects of P53 silencing and KRAS activation. Treatment of H3mut-P53/KRAS cells with low nicotine ECIG- and TCIG-conditioned media did not further enhance the degree of invasion observed in the untreated group.
We will next examine the effects of high nicotine conditioned media on cell invasion.

Essentially, they haven&#8217;t done enough to say if it will have direct effects on tumour invasion — but the cause for concern is what follows, in their description of changes to gene expression from the nicotine vapours alone.

Finally, gene expression studies show 263 differentially expressed genes following in vitro exposure to ECIG-conditioned media for 96hrs. The high nicotine ECIG-conditioned media induced a gene expression pattern similar to TCIG- conditioned media and whole cigarette smoke exposure in the H3mut-P53/KRAS cells. Preliminary analyses indicate the observed ECIG-specific gene expression changes were concordantly changed following TCIG-conditioned media exposure. We will next compare the ECIG-induced gene expression signature to carcinogenicity-related gene signatures established in previous and ongoing clinical investigations and test ECIG-altered candidate genes for their ability to drive the malignant transformation of airway epithelial cells. These studies will determine the impact of ECIG exposure on lung carcinogenicity and provide needed scientific guidance to the FDA regarding the physiologic effects of ECIGs.

There was a small comment in Nature around the finding, in which one of the authors made clear that it&#8217;s all very much preliminary work.

The changes are not identical, says study researcher Avrum Spira, who works on genomics and lung cancer at Boston University in Massachusetts. But “there are some striking similarities”, he says. The team is now evaluating whether the alterations mean that cells behave more like cancer cells in culture.
The work is at a very early stage and therefore cannot establish that e-cigarettes can cause cancer in vitro, let alone in vivo. “They may be safer [than tobacco], but our preliminary studies suggest that they may not be benign,” says Spira.
E-cigarettes are extremely controversial. Because they vaporize liquid containing nicotine, rather than burning tobacco, some researchers believe that the devices could greatly reduce the damage done to health by smoking; others, however, argue that they are simply ‘renormalizing’ smoking.

There&#8217;s a nice recent paper in Tobacco Control which has collated a bunch of results on e-cigarette emissions for anyone interested.

The delivery of nicotine and the release of tobacco-specific nitrosamines, aldehydes and metals are not consistent across products. Furthermore, the nicotine level listed on the labels of e-cigarette cartridges and refill solutions is often significantly different from measured values. Phenolic compounds, polycyclic aromatic hydrocarbons and drugs have also been reported in e-cigarette refill solutions, cartridges and aerosols. Varying results in particle size distributions of particular matter emissions from e-cigarettes across studies have been observed… Performance characteristics of e-cigarette devices also vary across and within brands.

The presence of the aldehydes (formaldehyde, acetaldehyde, acrolein, acetone) is pretty clear reason to believe these aren&#8217;t quite the clean-living alternative they&#8217;re cracked up to be.
Hopefully there are more in-depth reviews on the way, but at present these studies are few and far between.
⦿ Park et al. (2014) The effect of e-cigarette exposure on airway epithelial cell gene expression and transformation. Clin Cancer Res 20; B16⦿ Cheng (2014) Chemical evaluation of electronic cigarettes. Tob Control 23:ii11-ii17

E-cigarettes: what’s the harm ?

Tobacco smoking is now a globally accepted public health issue, and recently e-cigarettes have emerged as a socially acceptable form of an increasingly marginalised habit. They occupy a somewhat grey area, with those in medicine unsure whether to slate them if they might be a tool in getting hardcore smokers to quit the obviously more contaminated and harmful paper counterparts.

The BBC reports that e-cigarette users have tripled since 2010 (based on health charity Ash's estimates). The group say that use amongst non-smokers remains small (~1%).

NHS Choices gives a good impartial run-down of the organisation’s report, which highlights that roughly  ⅓ of e-cig users are ex- and ⅔ are current smokers. The NHS stresses that although this survey may capture the negligible rate of use among non-smokers now, it’s impossible to predict how use of such a new technology may develop.

Associate editor of the British Medical Journal Douglas Kamerow observed earlier this month that:

one can envisage three possible scenarios in which e-cigarettes are used: one good, two bad. Potentially, they could be useful to help tobacco smokers quit, replacing cigarettes and slowly dialing down nicotine levels, much as other nicotine delivery devices (gum, patches, inhalers, and so on) do. That would be a good thing, although the research that is starting to accumulate about this therapeutic use of e-cigarettes is so far not promising.

Secondly, e-cigarettes could be used to maintain a tobacco habit by allowing smokers nicotine intake in tobacco free areas, such as the workplace or home, because the vapor emitted from e-cigarettes is odorless and presumed to be not toxic. This is not so good for public health, as one of the most important incentives for quitting smoking has been the dramatic increase in places that prohibit it.

Thirdly, and most distressing, e-cigarettes could be used to initiate nicotine use and perhaps lead to tobacco smoking. With their many flavors and nicotine levels, they are easy to use and appealing to vapers of all ages, including adolescents. Friends report that an e-cigarette is the new status item in middle schools here, where they are easily concealed and used in bathrooms and outdoor breaks. Will this lead to an end or reversal of the long term downward trend in tobacco use? Time will tell. But the latest trend in e-cigarettes is perhaps more frightening still. Most early e-cigarettes were disposable appliances designed to look and feel like cigarettes; they even had a little LED light at the end that lit up when you inhaled. You used them up and then threw them away. What we’re seeing now, however, is larger vaporizing systems. Their batteries can be recharged with the USB port of your computer and the vaporizer refilled with nicotine liquid (called “juice”) in your choice of flavor at your preferred nicotine concentration.

The problem is thatnicotine is a poison, and the e-liquid is easily absorbed in several ways: inhalation, certainly, but also through ingestion and even skin contact. Add in the fact that the “juices” come in cute little bottles of colored, great smelling liquids and you have the makings of a terrific attraction for toddlers and young children. Unsurprisingly, US poison control centers are reporting threefold increases in calls about e-liquid poisonings. A slug of 36 mg/mL of e-liquid can make a child very sick. No deaths have been reported yet, but it is only a matter of time. Higher concentrations of e-liquid, available wholesale in gallon jugs on the internet for home brewing, are deadlier yet. And childrenaren’t theonlyvictims. Emergencyrooms are reporting adults showing up with cardiac symptoms after spilling e-liquid on their skin.

As a doctor, it must be hard to live through a generation that’s fought off the lawsuits and marketing ploys of Big Tobacco and its shifty lobbies even to just achieve scientific consensus on the harm smoking does, and then to see a 21st century version appear in its wake.

The New York Times provides further coverage of casualties in a society now keeping a toxic stimulant liquid within fragile containers.

Last month, a 2-year-old girl in Oklahoma City drank a small bottle of a parent’s nicotine liquid, started vomiting and was rushed to an emergency room.

That case and age group is considered typical. Of the 74 e-cigarette and nicotine poisoning cases called into Minnesota poison control in 2013, 29 involved children age 2 and under. In Oklahoma, all but two of the 25 cases in the first two months of this year involved children age 4 and under.

image

“This is one of the most potent naturally occurring toxins we have,” Mr. Cantrell said of nicotine. But e-liquids are now available almost everywhere. “It is sold all over the place. It is ubiquitous in society.”

The surge in poisonings reflects not only the growth of e-cigarettes but also a shift in technology. Initially, many e-cigarettes were disposable devices that looked like conventional cigarettes. Increasingly, however, they are larger, reusable gadgets that can be refilled with liquid, generally a combination of nicotine, flavorings and solvents. In Kentucky, where about 40 percent of cases involved adults, one woman was admitted to the hospital with cardiac problems after her e-cigarette broke in her bed, spilling the e-liquid, which was then absorbed through her skin.

The problems with adults, like those with children, owe to carelessness and lack of understanding of the risks. In the cases of exposure in children, “a lot of parents didn’t realize it was toxic until the kid started vomiting,” said Ashley Webb, director of the Kentucky Regional Poison Control Center at Kosair Children’s Hospital.

Children are often brought up as “passive” sufferers from cigarettes, but acute toxicity frames it quite starkly. Nicotine clearly has its dangers, but to put that aside for a moment, a study of the contaminants in e-cigarettes found nothing above workplace standards of toxicity.

Smoking has largely moved out of the media spotlight but its spectre looms large as the leading cause of cancer deaths in the world today

It’s been sidelined from film, television and advertising, and in many countries banned from indoor spaces. Responsible for 90% of all lung cancers, the most common of the world’s cancer, there remain too few indicators of who is at most risk (for lung cancer or COPD), and it’s still hazy as to why the risk lingers for individuals who stubbed out years ago.

Preclinical research using human bronchial cells containing mutations found in smokers at risk of lung cancer was presented recently at the American Association for Cancer Research annual meeting.

There has been very little experimental research into the subject at all but this posting describes the effect of e-cigarette exposure on airway epithelial cell gene expression and transformation, in the spirit of earlier papers which started to unpick the mutagenesis we’re now all so aware of.

The finding suggests that the e-cig is not as benign as presented.

The toxicity and potential carcinogenicity of ECIGs have not previously been evaluated. In this study, we assess the impact of ECIG exposure on the carcinogenic potential of immortalized human bronchial epithelial cells on a background of silenced p53 and activated KRAS (H3mut-P53/KRAS). This model is utilized because p53 and KRAS mutations are often observed in the airway of current and former smokers at risk for lung cancer.

For a guided tour of tumour suppressor p53’s mechanisms of inactivation, see last week’s post. KRAS is a similar gene, though works inversely by promoting hyperproliferation upon activation — what’s known as a proto-oncogene.

The epithelial cells were exposed to both a low and high concentration of nicotine in the ECIG vapor- or tobacco cigarette (TCIG) smoke-conditioned media. The lower nicotine concentration was selected to mimic the average plasma nicotine levels in electronic nicotine delivery system (ENDS) users and did not demonstrate toxic or anti-proliferative effects on the cells. The higher concentration was chosen to represent the anticipated nicotine levels to which the epithelial cells of smokers are actually exposed. In anchorage independent growth assays, the in vitro correlate of malignant transformation, we found enhanced colony growth in the H3mut-P53/KRAS cells following a 10-day treatment with the high nicotine ECIG- and TCIG-conditioned media compared to the untreated and low nicotine treatment groups.

We next assessed the effect of ECIG and TCIG exposure on cell invasion using a three-dimensional air-liquid interface (ALI) model. At baseline, H3mut-P53/KRAS cells exhibit invasive behavior in the ALI model, due to the downstream effects of P53 silencing and KRAS activation. Treatment of H3mut-P53/KRAS cells with low nicotine ECIG- and TCIG-conditioned media did not further enhance the degree of invasion observed in the untreated group.

We will next examine the effects of high nicotine conditioned media on cell invasion.

Essentially, they haven’t done enough to say if it will have direct effects on tumour invasion — but the cause for concern is what follows, in their description of changes to gene expression from the nicotine vapours alone.

Finally, gene expression studies show 263 differentially expressed genes following in vitro exposure to ECIG-conditioned media for 96hrs. The high nicotine ECIG-conditioned media induced a gene expression pattern similar to TCIG- conditioned media and whole cigarette smoke exposure in the H3mut-P53/KRAS cells. Preliminary analyses indicate the observed ECIG-specific gene expression changes were concordantly changed following TCIG-conditioned media exposure. We will next compare the ECIG-induced gene expression signature to carcinogenicity-related gene signatures established in previous and ongoing clinical investigations and test ECIG-altered candidate genes for their ability to drive the malignant transformation of airway epithelial cells. These studies will determine the impact of ECIG exposure on lung carcinogenicity and provide needed scientific guidance to the FDA regarding the physiologic effects of ECIGs.

There was a small comment in Nature around the finding, in which one of the authors made clear that it’s all very much preliminary work.

The changes are not identical, says study researcher Avrum Spira, who works on genomics and lung cancer at Boston University in Massachusetts. But “there are some striking similarities”, he says. The team is now evaluating whether the alterations mean that cells behave more like cancer cells in culture.

The work is at a very early stage and therefore cannot establish that e-cigarettes can cause cancer in vitro, let alone in vivo. “They may be safer [than tobacco], but our preliminary studies suggest that they may not be benign,” says Spira.

E-cigarettes are extremely controversial. Because they vaporize liquid containing nicotine, rather than burning tobacco, some researchers believe that the devices could greatly reduce the damage done to health by smoking; others, however, argue that they are simply ‘renormalizing’ smoking.

There’s a nice recent paper in Tobacco Control which has collated a bunch of results on e-cigarette emissions for anyone interested.

The delivery of nicotine and the release of tobacco-specific nitrosamines, aldehydes and metals are not consistent across products. Furthermore, the nicotine level listed on the labels of e-cigarette cartridges and refill solutions is often significantly different from measured values. Phenolic compounds, polycyclic aromatic hydrocarbons and drugs have also been reported in e-cigarette refill solutions, cartridges and aerosols. Varying results in particle size distributions of particular matter emissions from e-cigarettes across studies have been observed… Performance characteristics of e-cigarette devices also vary across and within brands.

The presence of the aldehydes (formaldehyde, acetaldehyde, acrolein, acetone) is pretty clear reason to believe these aren’t quite the clean-living alternative they’re cracked up to be.

Hopefully there are more in-depth reviews on the way, but at present these studies are few and far between.

⦿ Park et al. (2014) The effect of e-cigarette exposure on airway epithelial cell gene expression and transformationClin Cancer Res 20; B16

⦿ Cheng (2014) Chemical evaluation of electronic cigarettes. Tob Control 23:ii11-ii17

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Somewhat unexpectedly, this study also revealed that the density of TOM [Translocase of the Outer Membrane] clusters followed an inner-cellular gradient from the perinuclear to the peripheral mitochondria. Altogether, the reported findings showed a correlation of the metabolic activity of the cells and the nanoscale clustering of TOM. This suggests that the control of the distribution of TOM might be a mechanism to regulate protein import into mitochondria.

Interesting note on TOM from Jakobs and Wurm in Super-resolution microscopy of mitochondria, regarding a finding from a paper they wrote 3 years earlier.

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Super-resolution microscopy allows far better imaging of the organelles (as its name suggests) but equipment capable of 3D SR microscopy is limited to a small number of labs, meaning 2D is in wider use (compare ii and iii in the simulation); b-c on the diagram are simulations of the inner membrane, while d is a simulation of the outer mitochondrial membrane.

An outer membrane and a highly folded inner membrane constitute the intricate inner architecture of mitochondria. The invaginations of the inner membrane, called cristae, are not simply random wide infolds. Rather they are topologically complicated and their shape and number is adapted to the cellular requirements. The inner membrane hosts the oxidative phosphorylation system (OXPHOS).

Since the 1950s, various forms of electron microscopy (EM) have provided a detailed view on the membrane architecture of these organelles. EM is exquisitely suited for the investigation of membrane structures, but is generally less capable of determining the distribution of individual proteins. Typically, quantitative immunogold EM requires the decoration of sections with antibodies, resulting in relatively few gold particles per decorated section. To determine the suborganellar distribution of a specific protein with this approach, numerous individual gold localizations are recorded on many images and an average protein localization is determined. Hence immunogold EM is usually not suited to study protein distribution in individual mitochondria.

Fluorescence microscopy is arguably the most suitable approach to study the distribution of proteins in single mitochondria. However, studies using conventional fluorescence microscopy to investigate protein localizations in these organelles ultimately face the challenge that mitochondria are small; the width of mitochondrial tubules is typically between 250 and 500 nm. In conventional (confocal) microscopes diffraction limits the achievable resolution to ≥200 nm in the lateral plane and to ≥500 nm in the axial direction. Hence the size of most mitochondria is just at the resolution limit of optical microscopy making the analysis of submitochondrial protein distributions always challenging and often entirely impossible using diffraction limited optical microscopes.

Over the last decade several super-resolution microscopy (nanoscopy) concepts have been devised that allow diffraction-unlimited optical resolution. All concepts that fundamentally overcome the diffraction limit exploit a transition between two fluorophore states, usually a fluorescent (on-) and a non-fluorescent (off-) state in order to discriminate adjacent features. Depending on how the transition is implemented, the current super-resolution methods may be assigned to one of two classes, namely coordinate-targeted (prominent approaches: STED, SPEM/SSIM and RESOLFT and coordinate-stochastic approaches (PALM, STORM, FPALM, GSDIM, dSTORM, and others). The various methods routinely provide optical resolution well below 50 nm (i.e. they fundamentally overcome the diffraction barrier), have been implemented with more than one color, and 3D versions are available. The underlying physical concepts as well as the practical differences between the approaches have been expertly reviewed elsewhere.

I’ve only come across the TIMs before in any great detail (Tim9 and 10 specifically), but import of the small Tim proteins does not require the outer membrane’s TOM receptors (you may be able to spot Mia40 in the diagram below).

image

The group noted how TOM, while the main import pore for nuclear-encoded proteins into mitochondria, little is known about its spatial distribution within the outer membrane.

They used super-resolution stimulated emission depletion (STED) to determine a quantitative distribution of Tom20 (a subunit of the TOM complex) in over 1,000 cells. What they saw was the protein nestled in clusters, whose nanoscale distribution was “finely adjusted to the cellular growth conditions as well as to the specific position of a cell within a microcolony”.

The density of the clusters correlates to the mitochondrial membrane potential. The distributions of clusters of Tom20 and of Tom22 follow an inner-cellular gradient from the perinuclear to the peripheral mitochondria. We conclude that the nanoscale distribution of the TOM complex is finely adjusted to the cellular conditions, resulting in distribution gradients both within single cells and between adjacent cells.

This finding has been cited elsewhere just once to date, in a 2012 review Mitochondria: In sickness and in health.

The lateral organization of the OM is not as well understood, but it serves as a unique signaling platform for pathways such as BCL-2 protein-dependent apoptosis and innate antiviral immunity, which requires the regulated self-assembly of the mitochondrial localized membrane protein, MAVS, into a signaling complex essential for anti-inflammatory interferon response. Recent superresolution light microscopy techniques have revealed that the OM import TOM complex is localized in clusters, whose density and distribution are regulated by growth conditions that alter mitochondrial membrane potential.

This observation highlights that events inside mitochondria regulate the organization and activity of complexes at the mitochondrial surface, which can influence the external structure and behavior of the organelle.

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The external structure and the cellular location of mitochondria are critical for their function and depend on highly regulated activities such as mitochondrial division and fusion, motility, and tethering. These activities govern the overall shape, connectedness, and location of mitochondria within cells. Although little data are currently available, it is clear that the relative contributions of these activities and the molecular components that mediate them are highly tissue specific—a phenomenon that contributes to the variable manifestations of human mitochondrial diseases.

··· Jakobs and Wurm (2014) Super-resolution microscopy of mitochondria. Curr. Opin. Chem. Biol., 20:9–15

··· Nunnari and Suomalainen (2012) Mitochondria: in sickness and in health. Cell, 148(6), 1145-1159

··· Wurm et al. (2011) Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient. PNAS, 108, 13546–13551

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Above: non-enzymatic sugar phosphate interconversions in a plausible Archean ocean environment reproduce reactions from glycolysis and the pentose phosphate pathways – ‘central carbon metabolism’.
Origins of life on the ocean wave
I don&#8217;t think I&#8217;ve covered theories on biogenesis here before, but for anyone uninitiated on the topic, Larry Moran gave a good summary of the essentials over on his blog in 2009.

There are several competing hypotheses about the origin of life. Most people know about the Primordial Soup scenario; that&#8217;s the one where complex organic molecules are created by spontaneous chemical reactions. Over time these complex molecules, such as amino acids and nucleotides, accumulate in a warm little pond and eventually they come together to form proteins and nucleic acids.
 The RNA World scenario is similar except that nucleic acids (RNA) are thought to form before proteins. For a while, RNA molecules are the main catalysts in the primordial soup. Later on, proteins take over some of the catalytic roles. One of the problems with the RNA world hypothesis is that you have to have a reasonable concentration of nucleotides before the process can begin.
 The third hypothesis is called Metabolism First. In this scheme, the first reactions involve spontaneous formation of simple molecules such as acetate, a two-carbon compound formed from carbon dioxide and water. Pathways leading to the synthesis of simple organic molecules might be promoted by natural catalysts such as minerals and porous surfaces in rocks. The point is that the origin of life is triggered by the accumulation of very simple organic molecules in thermodynamically favorable circumstances.
 Simple organic molecules can then be combined in various ways that result in simple amino acids, lipids, etc. These, in turn, could act as catalysts for the formation of more organic molecules. This is the beginning of metabolism. 
 Eventually simple peptides will be formed and this could lead to better catalysts. Nucleic acids and complex amino acids will only form near the end of this process.

As Larry saw it back then, metabolism-first was the front-runner, offering a simple solution to the problem of life&#8217;s (homo)chirality. Other papers I&#8217;ve seen recently, and the work of Nick Lane (in press at PLOS) who gave a talk at my university last month likewise are in favour metabolism-first.
A new paper from authors split between Cambridge and Mill Hill (London) further bolsters the prospect of metabolism kicking off without enzymes, mimicking the chemical make-up of life&#8217;s earliest oceans in the lab. They found spontaneous occurrence of reaction sequences observed in modern organisms in the glycolysis and the pentose phosphate pathways ‒ ‘central carbon metabolism’.
What&#8217;s more, when iron was maintained ferrous Fe(II), the simulated chemistry of the anoxic Archean ocean stabilised the phosphorylated intermediates and accelerated intermediate reactions and production of pyruvate — a molecule at the intersection of multiple metabolic pathways.

The 29 consequently observed reactions (part c in the main figure above) include the formation of glucose, pyruvate, the nucleic acid precursor ribose-5-phosphate and the amino acid precursor erythrose-4-phosphate. Ferrous iron, Fe(II), is understood to have had high concentrations in the Archean oceans.

The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron-rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.

This is a really nice piece of work, building on earlier pieces that have found the core metabolic network is similar in all organisms. In 2000, Jeong and colleagues — including a couple of names I recognise - Albert and Barabasi — came to this conclusion in a paper on the large-scale organisation of metabolic networks on which I&#8217;ll be posting soon, and just last year Brakman and Smith wrote up a 62 page essay on The Logic of Metabolism which really was quite striking; one of those texts you can&#8217;t put down (seems such a long time ago now!).
» Phys.org: Was life inevitable? New paper pieces together metabolism&#8217;s beginnings» Open access arXiv preprint of the work: q-bio/1207.5532
All this just shows the authors know their field however, and from points made by others they build their investigation: are modern biochemical reaction sequences result of evolutionary selection, or was the initial metabolic reaction network a ready-made entity when life popped up?
As much as the tone is towards the latter, the authors concede both possibilities have their individual merit. Firm experimental evidence for either is sorely lacking.
Fundamental experiments
In water, the group observed 17 cases of glycolytic/pentose phosphate pathway (PPP) intermediates being converted into other metabolites.

In the pentose phosphate pathway, we observed isomerization of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, as well as the formation of glyceraldehyde 3-phosphate from these intermediates (Fig. A, left panel). In cellular metabolism, analogous reaction sequences do exist and are catalysed by the pentose phosphate pathway enzymes ribulose 5-phosphate epimerase, ribose 5-phosphate isomerase and transketolase (Fig. D, left panel). Glycolytic intermediates converted into pyruvate and glucose, the stable products of glycolysis and gluconeogenesis, as well as the intermediate metabolite glucose 6-phosphate (Fig. A, right panel).
Overall, pyruvate formation dominated. Its non-enzymatic formation was detected from PPP metabolites, fructose 6-phosphate, fructose 1,6-bisphosphate and all intermediates of lower glycolysis (Fig. D, right panel). It is therefore apparent that heat exposure is sufficient to convert intermediate metabolites of glycolysis and the pentose phosphate pathway into pyruvate and glucose that constitute thermodynamically stable products also in the modern, enzyme-catalysed metabolism, and to induce isomerization between pentose phosphate metabolites.

To clarify in case it may not be obvious having removed the figure caption, these diagrams show metabolic pathways, or ‘network topologies’ in plain old (high-purity ULC-MS grade) water (A), ‘plausible concentrations’ [for the Archean ocean] of Na, Cl, K, BO3, F, PO4, Mg, Ca, Si, Mo, Co, Ni and Fe (B), a ferrous Fe(II)-supplemented version of this Archean mimetic, i.e. maintaining anoxic conditions (C) and lastly the ‘modern’ enzyme-bolstered glycolysis (canonical Embden-Meyerhof) and pentose phosphate pathways.
After looking at water, the group tested whether conditions replicating the pre-oxygenation oceanic environment would influence these reactions, and as shown above they did, significantly. Amazingly (perhaps, though it does sound labour-intensive) they quantified the metabolites in the resultant 1,200 samples by manually supervised LC-MS/MS peak identification and integration of 18,000 resultant chromatographic peaks. Oceanic salts did not show any significant influence on the sugar phosphate interconversions, which is a notable result in itself.

However, a reactive solution containing the metal ions Fe, Co, Ni, Mo as well as phosphate at Archean ocean plausible concentrations catalysed additional reactions. In the metal-rich solution, out of the 182 reactions monitored, we observed in 28 cases the conversion of a glycolytic or pentose phosphate metabolite into another intermediate of the two pathways (Fig. B). The reactions catalysed by the Archean ocean mimetic yielded the formation of ribose 5-phosphate, the constituent of the RNA backbone and erythrose 4-phosphate, the precursor of aromatic amino acids, starting from several pentose phosphate pathway intermediates.
As a result, these non-enzymatic reactions interconnected all pentose phosphate pathway intermediates (glucose 6-phosphate, erythrose 4-phosphate, ribose 5-phosphate, ribulose 5-phosphate, xylulose 5-phosphate and sedoheptulose 7-phosphate). The interconversion network corresponds to the reaction sequence of the modern-cell non-oxidative pentose pathway (Fig. B and D). In difference, interconversion reactions that would correspond to reactions of the oxidative pentose phosphate pathway, converting glucose 6-phosphate to 6-phosphogluconate, were not observed.

This ocean-in-a-test-tube allowed some neat little tests to be carried out with 6-phosphogluconate (6PG), stable at 70°C in water, but reacting to form PPP intermediates (⇒ pyruvate). The sheer scale of these experiments is impressive ‒ they monitored 182 possible interconversion reactions in the iron-rich mimetic. How many trained lab monkeys does it take to watch 182 reactions you might ask — actually the team used LC-SRM, the second half of which stands for Selected Reaction Monitoring; another form of mass spectrometry. It saw some use in these experiments since it&#8217;s really sensitive (it can measure PTMs below the range of regular mass spec.) and its ‘data-independent nature’.
So as to give more than just a ‘network topology’, they were also able to show that ferrous iron increased specificity, leading to a greater mean carbon recovery in products (62% vs. 50%), i.e. ¼ of the absolute concentrations of non-enzymatically interconverted metabolites formed an intermediate of glycolysis/PPP (calculated with 62% being around ~125% of 50% in case you were wondering!) — i.e. there was quite a favourable yield in this ‘one-pot’ organic synthesis.

The determined reaction rates ranged over four orders of magnitude. In the ferrous iron-rich Archean ocean mimetic, the fastest observed reaction occurred at a rate 19.6 lM/h and was the conversion 6-phosphogluconate to the RNA backbone precursor ribose 5-phosphate. In contrast, the slowest reaction, the dephosphorylation of glucose 6-phosphate to glucose, occurred at a rate of 0.09 lM/h. Several of the interconversion rates were faster in the Archean ocean mimetics compared to the water (12 out of the 17 reactions detected in water); comparing the mimetics, 12 reactions were accelerated in the presence of Fe(II) over Fe(III). 6-phosphogluconate for instance was not converted to ribose 5-phosphate in water. In the ferric iron-rich mimetic, this reaction was observed at a rate of 5.5 lM/h, while in the ferrous iron-rich anoxic experiment at a rate of 19.6 lM/h. In some cases, acceleration was also observed for the three-carbon phosphate interconversion reactions that are analogous to reactions of lower glycolysis; that is, the conversion of phosphoenolpyruvate to pyruvate occurred at 13.5 lM/h in the Fe(II)-rich Archean ocean mimetic, while a rate of 7.3 lM/h was observed in water. However, several of the of three-carbon metabolite formation reactions, including the formation of pyruvate from fructose 1,6-bisphosphate, or glyceraldehyde 3-phosphate from ribose 5-phosphate, were slowed down, most likely due to the stabilization of precursors.
In order to get a global picture of the reactivity in the system, we therefore combined all intermediate metabolites at a concentration of 7.5 lM and determined the temperature-dependent rate of pyruvate formation. At temperatures of 40°C and below, no pyruvate was formed, confirming the absence of enzymatic contamination in the combined reaction mimetic. Above 50°C, pyruvate formation was detected and increased in a temperature-dependent manner. The fastest pyruvate formation rate was detected at 90°C, where typical metabolic enzymes are not functional. Comparing the three conditions, pyruvate formation rate was lowest in water, increased in the presence of the ocean components and achieved its highest rate in the ferrous iron-rich Archean ocean simulation. Compared to water at a temperature of 70°C, the pyruvate formation was 49% faster in the presence of the Archean ocean mimetic and ferric iron, and 200% faster in the analogous, ferrous iron containing ocean simulation. Hence, the ferrous condition plausible for the Archean ocean does not only catalyse interconversion among pentose phosphate sugars, they also favoured specificity and increased reactivity in a non-enzymatic, glycolysis-like, chemical reaction system.

The final step in proceedings set up a low concentration of the aforementioned mineral ions, with non-biological optimum temperatures ruling out any enzymes having snuck through, and what&#8217;s more confirming the ferrous iron increases reactivity of glycosysis-like chemical reactions.
What&#8217;s so fascinating about metabolism-first theories is that they still need evolutionary explanation ‒ that is, how did life come to harness these processes, and what did it bring to the table in doing so? Here, the authors note that the evolutionary origins of the Embden-Meyerhof network structure are ‘still largely unknown’.

Geological records reveal details about the chemical environment under which life spread for the first time. This event has been dated between the earliest Archean eon that followed the late heavy bombardment, likely between 4.1 and 3.5 billion years ago, when the first unequivocal traces of life have been dated. Geochemical and geological evidence, particularly the large iron isotope fractionations and the lack of sulphur isotope fractionation during pyrite burial, suggests that the oceans of this period were rich in ferrous iron and poor in sulfate. The absolute concentration of iron in these ancient oceans is equivocal with estimates ranging from 20 lM to 5 mM. Knowledge about the concentrations of other bio-essential metals in the Archean oceans during the evolution of life comes from reconstructing the biogeochemical cycles of these elements and taking into account the environmental properties of a world devoid of oxygen. This has led to the suggestion that copper concentrations in the Archean oceans were negligible and that cobalt and manganese were more abundant than today (but still many orders of magnitude lower than the paleo iron concentrations). The same argument has been applied to zinc; however, its concentration in Proterozoic black shales and Archean iron oxides suggests that its concentration may have remained closer to modern levels over much of the geological time. Nickel and molybdenum concentrations were likely lower than today, whereas concentrations of cobalt and manganese are assumed to be similar to modern levels. It has further been suggested that phosphorus is more easily released from sediment in anoxic (or dysoxic) bottom waters, hinting that the Archean oceans may have been rich in phosphate.

The only flaw in this paper is that in parts it feels a little vague ‒ I don&#8217;t think I&#8217;ll be the only person reading this wondering exactly what the significance of comparing network topology is, but it could well be my own miseducation showing through. Similarly, the how of the origin of life is sparse on detail, but in fairness this is asking a bit much of one paper.

One of the difficulties in describing the origin of metabolism is the fact that the metabolic network is largely composed of intermediates that are not characterized by long-time stability, at least when considering geological environments and timescales. As shown here and previously, this in particular applies to sugar phosphate molecules. In addition, large sugar phosphates are not frequently generated in experiments that address scenarios of primordial carbon fixation. This difficulty cannot, however, mask the fact that sugar phosphates are constituents of many molecules, such as RNA, DNA, ATP and lipids, which are inevitably connected with the emergence of life. It is the fundamental role of sugar phosphates, and the virtual universality of their few metabolic interconversion sequences, that places their origin to the very early evolutionary stages.
Long-term stability seems thus not to be a predictor of whether a molecule adopts a cellular metabolic function. A possible explanation is that molecules stable in a certain environment do not react with their surrounding molecules; inert molecules were thus unlikely to form reaction systems based on environmental catalysts. This seeming paradox of the universality of sugar phosphates and their low stability in the prebiotic world might be solved by accepting different reaction sequences for carbon fixation and the first forms of metabolism: Carbon fixation could have occurred through non-metabolism-like events, including the so-called formose reaction, which is, in a series of condensation steps, able to convert several formaldehyde molecules into complex carbohydrates structures, or through alternative/parallel scenarios that include mineral- or photochemically catalysed reactions, as well as microcompartmentalization, that allowed the accumulation of first biomolecules in protocells. It is only when the first biomolecules had achieved life-compatible concentrations that biologically relevant interconversion sequences, or early forms of metabolic pathways, could become active.

This seems to place their previous results in a bit of a grey area, given that they were produced with the intention of simulating environmental conditions, but in discussing their ideas on the matter the authors are pointing elsewhere for the first sparks of metabolism. The team are the first to point out what they missed — mineral surfaces can act as catalysts; reaction conditions were not optimised to maximise the number of reactions (rather, the reconstruction ran the other way around).

It is thus equally well possible that this part of the pathway came into being with the emergence of enzymes. Therefore, these results could support a hybrid hypothesis to describe the origin of metabolism: a core set of reactions would have be constrained by the environment of the early world organisms, and this network was then extended in terms of both reactivity and efficiency through the evolutionary selection of enzymes until the modern network was in place.

The array of possibilities is staggering. Lastly, it wouldn&#8217;t be an origin of life story without some weird Creature From The Deep, and before finishing the group slip in mention of thermophilic microorganisms that have a functional ‘non-phosphorylating’ Entner-Doudoroff pathway, not yielding any ATP from glucose, just metabolic intermediates. This gives some hope to the possibility of non-ATP-generating glycolysis as described having likewise been a part of the grand scheme of things back in some long-bygone era.

The results presented here demonstrate that sugar phosphate interconversions reactions are prebiotically plausible; thus, the origin of the ribose 5-phosphate through a sugar phosphate interconversion route should be considered. In this context, we have noticed that ribose 5-phosphate was around five times more stable than the other pentose phosphate intermediates (xylulose 5-phosphate and ribulose 5-phosphate) and the formation of ribose 5-phosphate from 6-phosphogluconate was the fastest of all reactions in the presence of Fe(II) (Fig 5A). These properties could have contributed to the central importance of ribose 5-phosphate as backbone of the genetic material.

Looking ahead, the authors propose a deeper inspection of the thermodynamics here, which I look forward to seeing and will fit in nicely with others in the field working towards the same goal.

Studies about the thermodynamics in metabolic systems have, however, shown that reaction equilibrium is shifted under conditions that separate a product from the catalyst, when reactions are coupled and when one product is stabilized, that is, by the inclusion in protocell-like vesicles (Amend et al, 2013). In future studies, it should thus be investigated whether the reactions reported here can be combined with prebiotically plausible mechanisms to stabilize the higher energetic phosphates and thus whether the reactions can be exploited in carbon fixation mechanisms as well.

Update: Larry has now written about this paper himself, and is discussing how he feels it failed to raise contradictions with alternative theories in the comments with co-author Markus Ralser. Interestingly he classifies this as the ‘metabolic soup’ line of thinking, since gluconeogenesis would have sprung up before glycolysis and pentose phosphate pathways.
In particular he seems to feel the authors failed to cite other research (I mean even I knew Nick Lane is one of the eminent scholars in this area yet his work is only cited once, in passing). Food for thought, and a reminder to consider what might be going unsaid. I&#8217;ll have to check back after Ralser has replied, could be an interesting discussion.
⇅ Keller MA, Turchyn AV and Ralser M (2014) Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in the plausible Archean ocean. Molecular Systems Biology 10:725
Above: non-enzymatic sugar phosphate interconversions in a plausible Archean ocean environment reproduce reactions from glycolysis and the pentose phosphate pathways – ‘central carbon metabolism’.
Origins of life on the ocean wave
I don&#8217;t think I&#8217;ve covered theories on biogenesis here before, but for anyone uninitiated on the topic, Larry Moran gave a good summary of the essentials over on his blog in 2009.

There are several competing hypotheses about the origin of life. Most people know about the Primordial Soup scenario; that&#8217;s the one where complex organic molecules are created by spontaneous chemical reactions. Over time these complex molecules, such as amino acids and nucleotides, accumulate in a warm little pond and eventually they come together to form proteins and nucleic acids.
 The RNA World scenario is similar except that nucleic acids (RNA) are thought to form before proteins. For a while, RNA molecules are the main catalysts in the primordial soup. Later on, proteins take over some of the catalytic roles. One of the problems with the RNA world hypothesis is that you have to have a reasonable concentration of nucleotides before the process can begin.
 The third hypothesis is called Metabolism First. In this scheme, the first reactions involve spontaneous formation of simple molecules such as acetate, a two-carbon compound formed from carbon dioxide and water. Pathways leading to the synthesis of simple organic molecules might be promoted by natural catalysts such as minerals and porous surfaces in rocks. The point is that the origin of life is triggered by the accumulation of very simple organic molecules in thermodynamically favorable circumstances.
 Simple organic molecules can then be combined in various ways that result in simple amino acids, lipids, etc. These, in turn, could act as catalysts for the formation of more organic molecules. This is the beginning of metabolism. 
 Eventually simple peptides will be formed and this could lead to better catalysts. Nucleic acids and complex amino acids will only form near the end of this process.

As Larry saw it back then, metabolism-first was the front-runner, offering a simple solution to the problem of life&#8217;s (homo)chirality. Other papers I&#8217;ve seen recently, and the work of Nick Lane (in press at PLOS) who gave a talk at my university last month likewise are in favour metabolism-first.
A new paper from authors split between Cambridge and Mill Hill (London) further bolsters the prospect of metabolism kicking off without enzymes, mimicking the chemical make-up of life&#8217;s earliest oceans in the lab. They found spontaneous occurrence of reaction sequences observed in modern organisms in the glycolysis and the pentose phosphate pathways ‒ ‘central carbon metabolism’.
What&#8217;s more, when iron was maintained ferrous Fe(II), the simulated chemistry of the anoxic Archean ocean stabilised the phosphorylated intermediates and accelerated intermediate reactions and production of pyruvate — a molecule at the intersection of multiple metabolic pathways.

The 29 consequently observed reactions (part c in the main figure above) include the formation of glucose, pyruvate, the nucleic acid precursor ribose-5-phosphate and the amino acid precursor erythrose-4-phosphate. Ferrous iron, Fe(II), is understood to have had high concentrations in the Archean oceans.

The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron-rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.

This is a really nice piece of work, building on earlier pieces that have found the core metabolic network is similar in all organisms. In 2000, Jeong and colleagues — including a couple of names I recognise - Albert and Barabasi — came to this conclusion in a paper on the large-scale organisation of metabolic networks on which I&#8217;ll be posting soon, and just last year Brakman and Smith wrote up a 62 page essay on The Logic of Metabolism which really was quite striking; one of those texts you can&#8217;t put down (seems such a long time ago now!).
» Phys.org: Was life inevitable? New paper pieces together metabolism&#8217;s beginnings» Open access arXiv preprint of the work: q-bio/1207.5532
All this just shows the authors know their field however, and from points made by others they build their investigation: are modern biochemical reaction sequences result of evolutionary selection, or was the initial metabolic reaction network a ready-made entity when life popped up?
As much as the tone is towards the latter, the authors concede both possibilities have their individual merit. Firm experimental evidence for either is sorely lacking.
Fundamental experiments
In water, the group observed 17 cases of glycolytic/pentose phosphate pathway (PPP) intermediates being converted into other metabolites.

In the pentose phosphate pathway, we observed isomerization of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, as well as the formation of glyceraldehyde 3-phosphate from these intermediates (Fig. A, left panel). In cellular metabolism, analogous reaction sequences do exist and are catalysed by the pentose phosphate pathway enzymes ribulose 5-phosphate epimerase, ribose 5-phosphate isomerase and transketolase (Fig. D, left panel). Glycolytic intermediates converted into pyruvate and glucose, the stable products of glycolysis and gluconeogenesis, as well as the intermediate metabolite glucose 6-phosphate (Fig. A, right panel).
Overall, pyruvate formation dominated. Its non-enzymatic formation was detected from PPP metabolites, fructose 6-phosphate, fructose 1,6-bisphosphate and all intermediates of lower glycolysis (Fig. D, right panel). It is therefore apparent that heat exposure is sufficient to convert intermediate metabolites of glycolysis and the pentose phosphate pathway into pyruvate and glucose that constitute thermodynamically stable products also in the modern, enzyme-catalysed metabolism, and to induce isomerization between pentose phosphate metabolites.

To clarify in case it may not be obvious having removed the figure caption, these diagrams show metabolic pathways, or ‘network topologies’ in plain old (high-purity ULC-MS grade) water (A), ‘plausible concentrations’ [for the Archean ocean] of Na, Cl, K, BO3, F, PO4, Mg, Ca, Si, Mo, Co, Ni and Fe (B), a ferrous Fe(II)-supplemented version of this Archean mimetic, i.e. maintaining anoxic conditions (C) and lastly the ‘modern’ enzyme-bolstered glycolysis (canonical Embden-Meyerhof) and pentose phosphate pathways.
After looking at water, the group tested whether conditions replicating the pre-oxygenation oceanic environment would influence these reactions, and as shown above they did, significantly. Amazingly (perhaps, though it does sound labour-intensive) they quantified the metabolites in the resultant 1,200 samples by manually supervised LC-MS/MS peak identification and integration of 18,000 resultant chromatographic peaks. Oceanic salts did not show any significant influence on the sugar phosphate interconversions, which is a notable result in itself.

However, a reactive solution containing the metal ions Fe, Co, Ni, Mo as well as phosphate at Archean ocean plausible concentrations catalysed additional reactions. In the metal-rich solution, out of the 182 reactions monitored, we observed in 28 cases the conversion of a glycolytic or pentose phosphate metabolite into another intermediate of the two pathways (Fig. B). The reactions catalysed by the Archean ocean mimetic yielded the formation of ribose 5-phosphate, the constituent of the RNA backbone and erythrose 4-phosphate, the precursor of aromatic amino acids, starting from several pentose phosphate pathway intermediates.
As a result, these non-enzymatic reactions interconnected all pentose phosphate pathway intermediates (glucose 6-phosphate, erythrose 4-phosphate, ribose 5-phosphate, ribulose 5-phosphate, xylulose 5-phosphate and sedoheptulose 7-phosphate). The interconversion network corresponds to the reaction sequence of the modern-cell non-oxidative pentose pathway (Fig. B and D). In difference, interconversion reactions that would correspond to reactions of the oxidative pentose phosphate pathway, converting glucose 6-phosphate to 6-phosphogluconate, were not observed.

This ocean-in-a-test-tube allowed some neat little tests to be carried out with 6-phosphogluconate (6PG), stable at 70°C in water, but reacting to form PPP intermediates (⇒ pyruvate). The sheer scale of these experiments is impressive ‒ they monitored 182 possible interconversion reactions in the iron-rich mimetic. How many trained lab monkeys does it take to watch 182 reactions you might ask — actually the team used LC-SRM, the second half of which stands for Selected Reaction Monitoring; another form of mass spectrometry. It saw some use in these experiments since it&#8217;s really sensitive (it can measure PTMs below the range of regular mass spec.) and its ‘data-independent nature’.
So as to give more than just a ‘network topology’, they were also able to show that ferrous iron increased specificity, leading to a greater mean carbon recovery in products (62% vs. 50%), i.e. ¼ of the absolute concentrations of non-enzymatically interconverted metabolites formed an intermediate of glycolysis/PPP (calculated with 62% being around ~125% of 50% in case you were wondering!) — i.e. there was quite a favourable yield in this ‘one-pot’ organic synthesis.

The determined reaction rates ranged over four orders of magnitude. In the ferrous iron-rich Archean ocean mimetic, the fastest observed reaction occurred at a rate 19.6 lM/h and was the conversion 6-phosphogluconate to the RNA backbone precursor ribose 5-phosphate. In contrast, the slowest reaction, the dephosphorylation of glucose 6-phosphate to glucose, occurred at a rate of 0.09 lM/h. Several of the interconversion rates were faster in the Archean ocean mimetics compared to the water (12 out of the 17 reactions detected in water); comparing the mimetics, 12 reactions were accelerated in the presence of Fe(II) over Fe(III). 6-phosphogluconate for instance was not converted to ribose 5-phosphate in water. In the ferric iron-rich mimetic, this reaction was observed at a rate of 5.5 lM/h, while in the ferrous iron-rich anoxic experiment at a rate of 19.6 lM/h. In some cases, acceleration was also observed for the three-carbon phosphate interconversion reactions that are analogous to reactions of lower glycolysis; that is, the conversion of phosphoenolpyruvate to pyruvate occurred at 13.5 lM/h in the Fe(II)-rich Archean ocean mimetic, while a rate of 7.3 lM/h was observed in water. However, several of the of three-carbon metabolite formation reactions, including the formation of pyruvate from fructose 1,6-bisphosphate, or glyceraldehyde 3-phosphate from ribose 5-phosphate, were slowed down, most likely due to the stabilization of precursors.
In order to get a global picture of the reactivity in the system, we therefore combined all intermediate metabolites at a concentration of 7.5 lM and determined the temperature-dependent rate of pyruvate formation. At temperatures of 40°C and below, no pyruvate was formed, confirming the absence of enzymatic contamination in the combined reaction mimetic. Above 50°C, pyruvate formation was detected and increased in a temperature-dependent manner. The fastest pyruvate formation rate was detected at 90°C, where typical metabolic enzymes are not functional. Comparing the three conditions, pyruvate formation rate was lowest in water, increased in the presence of the ocean components and achieved its highest rate in the ferrous iron-rich Archean ocean simulation. Compared to water at a temperature of 70°C, the pyruvate formation was 49% faster in the presence of the Archean ocean mimetic and ferric iron, and 200% faster in the analogous, ferrous iron containing ocean simulation. Hence, the ferrous condition plausible for the Archean ocean does not only catalyse interconversion among pentose phosphate sugars, they also favoured specificity and increased reactivity in a non-enzymatic, glycolysis-like, chemical reaction system.

The final step in proceedings set up a low concentration of the aforementioned mineral ions, with non-biological optimum temperatures ruling out any enzymes having snuck through, and what&#8217;s more confirming the ferrous iron increases reactivity of glycosysis-like chemical reactions.
What&#8217;s so fascinating about metabolism-first theories is that they still need evolutionary explanation ‒ that is, how did life come to harness these processes, and what did it bring to the table in doing so? Here, the authors note that the evolutionary origins of the Embden-Meyerhof network structure are ‘still largely unknown’.

Geological records reveal details about the chemical environment under which life spread for the first time. This event has been dated between the earliest Archean eon that followed the late heavy bombardment, likely between 4.1 and 3.5 billion years ago, when the first unequivocal traces of life have been dated. Geochemical and geological evidence, particularly the large iron isotope fractionations and the lack of sulphur isotope fractionation during pyrite burial, suggests that the oceans of this period were rich in ferrous iron and poor in sulfate. The absolute concentration of iron in these ancient oceans is equivocal with estimates ranging from 20 lM to 5 mM. Knowledge about the concentrations of other bio-essential metals in the Archean oceans during the evolution of life comes from reconstructing the biogeochemical cycles of these elements and taking into account the environmental properties of a world devoid of oxygen. This has led to the suggestion that copper concentrations in the Archean oceans were negligible and that cobalt and manganese were more abundant than today (but still many orders of magnitude lower than the paleo iron concentrations). The same argument has been applied to zinc; however, its concentration in Proterozoic black shales and Archean iron oxides suggests that its concentration may have remained closer to modern levels over much of the geological time. Nickel and molybdenum concentrations were likely lower than today, whereas concentrations of cobalt and manganese are assumed to be similar to modern levels. It has further been suggested that phosphorus is more easily released from sediment in anoxic (or dysoxic) bottom waters, hinting that the Archean oceans may have been rich in phosphate.

The only flaw in this paper is that in parts it feels a little vague ‒ I don&#8217;t think I&#8217;ll be the only person reading this wondering exactly what the significance of comparing network topology is, but it could well be my own miseducation showing through. Similarly, the how of the origin of life is sparse on detail, but in fairness this is asking a bit much of one paper.

One of the difficulties in describing the origin of metabolism is the fact that the metabolic network is largely composed of intermediates that are not characterized by long-time stability, at least when considering geological environments and timescales. As shown here and previously, this in particular applies to sugar phosphate molecules. In addition, large sugar phosphates are not frequently generated in experiments that address scenarios of primordial carbon fixation. This difficulty cannot, however, mask the fact that sugar phosphates are constituents of many molecules, such as RNA, DNA, ATP and lipids, which are inevitably connected with the emergence of life. It is the fundamental role of sugar phosphates, and the virtual universality of their few metabolic interconversion sequences, that places their origin to the very early evolutionary stages.
Long-term stability seems thus not to be a predictor of whether a molecule adopts a cellular metabolic function. A possible explanation is that molecules stable in a certain environment do not react with their surrounding molecules; inert molecules were thus unlikely to form reaction systems based on environmental catalysts. This seeming paradox of the universality of sugar phosphates and their low stability in the prebiotic world might be solved by accepting different reaction sequences for carbon fixation and the first forms of metabolism: Carbon fixation could have occurred through non-metabolism-like events, including the so-called formose reaction, which is, in a series of condensation steps, able to convert several formaldehyde molecules into complex carbohydrates structures, or through alternative/parallel scenarios that include mineral- or photochemically catalysed reactions, as well as microcompartmentalization, that allowed the accumulation of first biomolecules in protocells. It is only when the first biomolecules had achieved life-compatible concentrations that biologically relevant interconversion sequences, or early forms of metabolic pathways, could become active.

This seems to place their previous results in a bit of a grey area, given that they were produced with the intention of simulating environmental conditions, but in discussing their ideas on the matter the authors are pointing elsewhere for the first sparks of metabolism. The team are the first to point out what they missed — mineral surfaces can act as catalysts; reaction conditions were not optimised to maximise the number of reactions (rather, the reconstruction ran the other way around).

It is thus equally well possible that this part of the pathway came into being with the emergence of enzymes. Therefore, these results could support a hybrid hypothesis to describe the origin of metabolism: a core set of reactions would have be constrained by the environment of the early world organisms, and this network was then extended in terms of both reactivity and efficiency through the evolutionary selection of enzymes until the modern network was in place.

The array of possibilities is staggering. Lastly, it wouldn&#8217;t be an origin of life story without some weird Creature From The Deep, and before finishing the group slip in mention of thermophilic microorganisms that have a functional ‘non-phosphorylating’ Entner-Doudoroff pathway, not yielding any ATP from glucose, just metabolic intermediates. This gives some hope to the possibility of non-ATP-generating glycolysis as described having likewise been a part of the grand scheme of things back in some long-bygone era.

The results presented here demonstrate that sugar phosphate interconversions reactions are prebiotically plausible; thus, the origin of the ribose 5-phosphate through a sugar phosphate interconversion route should be considered. In this context, we have noticed that ribose 5-phosphate was around five times more stable than the other pentose phosphate intermediates (xylulose 5-phosphate and ribulose 5-phosphate) and the formation of ribose 5-phosphate from 6-phosphogluconate was the fastest of all reactions in the presence of Fe(II) (Fig 5A). These properties could have contributed to the central importance of ribose 5-phosphate as backbone of the genetic material.

Looking ahead, the authors propose a deeper inspection of the thermodynamics here, which I look forward to seeing and will fit in nicely with others in the field working towards the same goal.

Studies about the thermodynamics in metabolic systems have, however, shown that reaction equilibrium is shifted under conditions that separate a product from the catalyst, when reactions are coupled and when one product is stabilized, that is, by the inclusion in protocell-like vesicles (Amend et al, 2013). In future studies, it should thus be investigated whether the reactions reported here can be combined with prebiotically plausible mechanisms to stabilize the higher energetic phosphates and thus whether the reactions can be exploited in carbon fixation mechanisms as well.

Update: Larry has now written about this paper himself, and is discussing how he feels it failed to raise contradictions with alternative theories in the comments with co-author Markus Ralser. Interestingly he classifies this as the ‘metabolic soup’ line of thinking, since gluconeogenesis would have sprung up before glycolysis and pentose phosphate pathways.
In particular he seems to feel the authors failed to cite other research (I mean even I knew Nick Lane is one of the eminent scholars in this area yet his work is only cited once, in passing). Food for thought, and a reminder to consider what might be going unsaid. I&#8217;ll have to check back after Ralser has replied, could be an interesting discussion.
⇅ Keller MA, Turchyn AV and Ralser M (2014) Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in the plausible Archean ocean. Molecular Systems Biology 10:725
Above: non-enzymatic sugar phosphate interconversions in a plausible Archean ocean environment reproduce reactions from glycolysis and the pentose phosphate pathways – ‘central carbon metabolism’.
Origins of life on the ocean wave
I don&#8217;t think I&#8217;ve covered theories on biogenesis here before, but for anyone uninitiated on the topic, Larry Moran gave a good summary of the essentials over on his blog in 2009.

There are several competing hypotheses about the origin of life. Most people know about the Primordial Soup scenario; that&#8217;s the one where complex organic molecules are created by spontaneous chemical reactions. Over time these complex molecules, such as amino acids and nucleotides, accumulate in a warm little pond and eventually they come together to form proteins and nucleic acids.
 The RNA World scenario is similar except that nucleic acids (RNA) are thought to form before proteins. For a while, RNA molecules are the main catalysts in the primordial soup. Later on, proteins take over some of the catalytic roles. One of the problems with the RNA world hypothesis is that you have to have a reasonable concentration of nucleotides before the process can begin.
 The third hypothesis is called Metabolism First. In this scheme, the first reactions involve spontaneous formation of simple molecules such as acetate, a two-carbon compound formed from carbon dioxide and water. Pathways leading to the synthesis of simple organic molecules might be promoted by natural catalysts such as minerals and porous surfaces in rocks. The point is that the origin of life is triggered by the accumulation of very simple organic molecules in thermodynamically favorable circumstances.
 Simple organic molecules can then be combined in various ways that result in simple amino acids, lipids, etc. These, in turn, could act as catalysts for the formation of more organic molecules. This is the beginning of metabolism. 
 Eventually simple peptides will be formed and this could lead to better catalysts. Nucleic acids and complex amino acids will only form near the end of this process.

As Larry saw it back then, metabolism-first was the front-runner, offering a simple solution to the problem of life&#8217;s (homo)chirality. Other papers I&#8217;ve seen recently, and the work of Nick Lane (in press at PLOS) who gave a talk at my university last month likewise are in favour metabolism-first.
A new paper from authors split between Cambridge and Mill Hill (London) further bolsters the prospect of metabolism kicking off without enzymes, mimicking the chemical make-up of life&#8217;s earliest oceans in the lab. They found spontaneous occurrence of reaction sequences observed in modern organisms in the glycolysis and the pentose phosphate pathways ‒ ‘central carbon metabolism’.
What&#8217;s more, when iron was maintained ferrous Fe(II), the simulated chemistry of the anoxic Archean ocean stabilised the phosphorylated intermediates and accelerated intermediate reactions and production of pyruvate — a molecule at the intersection of multiple metabolic pathways.

The 29 consequently observed reactions (part c in the main figure above) include the formation of glucose, pyruvate, the nucleic acid precursor ribose-5-phosphate and the amino acid precursor erythrose-4-phosphate. Ferrous iron, Fe(II), is understood to have had high concentrations in the Archean oceans.

The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron-rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.

This is a really nice piece of work, building on earlier pieces that have found the core metabolic network is similar in all organisms. In 2000, Jeong and colleagues — including a couple of names I recognise - Albert and Barabasi — came to this conclusion in a paper on the large-scale organisation of metabolic networks on which I&#8217;ll be posting soon, and just last year Brakman and Smith wrote up a 62 page essay on The Logic of Metabolism which really was quite striking; one of those texts you can&#8217;t put down (seems such a long time ago now!).
» Phys.org: Was life inevitable? New paper pieces together metabolism&#8217;s beginnings» Open access arXiv preprint of the work: q-bio/1207.5532
All this just shows the authors know their field however, and from points made by others they build their investigation: are modern biochemical reaction sequences result of evolutionary selection, or was the initial metabolic reaction network a ready-made entity when life popped up?
As much as the tone is towards the latter, the authors concede both possibilities have their individual merit. Firm experimental evidence for either is sorely lacking.
Fundamental experiments
In water, the group observed 17 cases of glycolytic/pentose phosphate pathway (PPP) intermediates being converted into other metabolites.

In the pentose phosphate pathway, we observed isomerization of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, as well as the formation of glyceraldehyde 3-phosphate from these intermediates (Fig. A, left panel). In cellular metabolism, analogous reaction sequences do exist and are catalysed by the pentose phosphate pathway enzymes ribulose 5-phosphate epimerase, ribose 5-phosphate isomerase and transketolase (Fig. D, left panel). Glycolytic intermediates converted into pyruvate and glucose, the stable products of glycolysis and gluconeogenesis, as well as the intermediate metabolite glucose 6-phosphate (Fig. A, right panel).
Overall, pyruvate formation dominated. Its non-enzymatic formation was detected from PPP metabolites, fructose 6-phosphate, fructose 1,6-bisphosphate and all intermediates of lower glycolysis (Fig. D, right panel). It is therefore apparent that heat exposure is sufficient to convert intermediate metabolites of glycolysis and the pentose phosphate pathway into pyruvate and glucose that constitute thermodynamically stable products also in the modern, enzyme-catalysed metabolism, and to induce isomerization between pentose phosphate metabolites.

To clarify in case it may not be obvious having removed the figure caption, these diagrams show metabolic pathways, or ‘network topologies’ in plain old (high-purity ULC-MS grade) water (A), ‘plausible concentrations’ [for the Archean ocean] of Na, Cl, K, BO3, F, PO4, Mg, Ca, Si, Mo, Co, Ni and Fe (B), a ferrous Fe(II)-supplemented version of this Archean mimetic, i.e. maintaining anoxic conditions (C) and lastly the ‘modern’ enzyme-bolstered glycolysis (canonical Embden-Meyerhof) and pentose phosphate pathways.
After looking at water, the group tested whether conditions replicating the pre-oxygenation oceanic environment would influence these reactions, and as shown above they did, significantly. Amazingly (perhaps, though it does sound labour-intensive) they quantified the metabolites in the resultant 1,200 samples by manually supervised LC-MS/MS peak identification and integration of 18,000 resultant chromatographic peaks. Oceanic salts did not show any significant influence on the sugar phosphate interconversions, which is a notable result in itself.

However, a reactive solution containing the metal ions Fe, Co, Ni, Mo as well as phosphate at Archean ocean plausible concentrations catalysed additional reactions. In the metal-rich solution, out of the 182 reactions monitored, we observed in 28 cases the conversion of a glycolytic or pentose phosphate metabolite into another intermediate of the two pathways (Fig. B). The reactions catalysed by the Archean ocean mimetic yielded the formation of ribose 5-phosphate, the constituent of the RNA backbone and erythrose 4-phosphate, the precursor of aromatic amino acids, starting from several pentose phosphate pathway intermediates.
As a result, these non-enzymatic reactions interconnected all pentose phosphate pathway intermediates (glucose 6-phosphate, erythrose 4-phosphate, ribose 5-phosphate, ribulose 5-phosphate, xylulose 5-phosphate and sedoheptulose 7-phosphate). The interconversion network corresponds to the reaction sequence of the modern-cell non-oxidative pentose pathway (Fig. B and D). In difference, interconversion reactions that would correspond to reactions of the oxidative pentose phosphate pathway, converting glucose 6-phosphate to 6-phosphogluconate, were not observed.

This ocean-in-a-test-tube allowed some neat little tests to be carried out with 6-phosphogluconate (6PG), stable at 70°C in water, but reacting to form PPP intermediates (⇒ pyruvate). The sheer scale of these experiments is impressive ‒ they monitored 182 possible interconversion reactions in the iron-rich mimetic. How many trained lab monkeys does it take to watch 182 reactions you might ask — actually the team used LC-SRM, the second half of which stands for Selected Reaction Monitoring; another form of mass spectrometry. It saw some use in these experiments since it&#8217;s really sensitive (it can measure PTMs below the range of regular mass spec.) and its ‘data-independent nature’.
So as to give more than just a ‘network topology’, they were also able to show that ferrous iron increased specificity, leading to a greater mean carbon recovery in products (62% vs. 50%), i.e. ¼ of the absolute concentrations of non-enzymatically interconverted metabolites formed an intermediate of glycolysis/PPP (calculated with 62% being around ~125% of 50% in case you were wondering!) — i.e. there was quite a favourable yield in this ‘one-pot’ organic synthesis.

The determined reaction rates ranged over four orders of magnitude. In the ferrous iron-rich Archean ocean mimetic, the fastest observed reaction occurred at a rate 19.6 lM/h and was the conversion 6-phosphogluconate to the RNA backbone precursor ribose 5-phosphate. In contrast, the slowest reaction, the dephosphorylation of glucose 6-phosphate to glucose, occurred at a rate of 0.09 lM/h. Several of the interconversion rates were faster in the Archean ocean mimetics compared to the water (12 out of the 17 reactions detected in water); comparing the mimetics, 12 reactions were accelerated in the presence of Fe(II) over Fe(III). 6-phosphogluconate for instance was not converted to ribose 5-phosphate in water. In the ferric iron-rich mimetic, this reaction was observed at a rate of 5.5 lM/h, while in the ferrous iron-rich anoxic experiment at a rate of 19.6 lM/h. In some cases, acceleration was also observed for the three-carbon phosphate interconversion reactions that are analogous to reactions of lower glycolysis; that is, the conversion of phosphoenolpyruvate to pyruvate occurred at 13.5 lM/h in the Fe(II)-rich Archean ocean mimetic, while a rate of 7.3 lM/h was observed in water. However, several of the of three-carbon metabolite formation reactions, including the formation of pyruvate from fructose 1,6-bisphosphate, or glyceraldehyde 3-phosphate from ribose 5-phosphate, were slowed down, most likely due to the stabilization of precursors.
In order to get a global picture of the reactivity in the system, we therefore combined all intermediate metabolites at a concentration of 7.5 lM and determined the temperature-dependent rate of pyruvate formation. At temperatures of 40°C and below, no pyruvate was formed, confirming the absence of enzymatic contamination in the combined reaction mimetic. Above 50°C, pyruvate formation was detected and increased in a temperature-dependent manner. The fastest pyruvate formation rate was detected at 90°C, where typical metabolic enzymes are not functional. Comparing the three conditions, pyruvate formation rate was lowest in water, increased in the presence of the ocean components and achieved its highest rate in the ferrous iron-rich Archean ocean simulation. Compared to water at a temperature of 70°C, the pyruvate formation was 49% faster in the presence of the Archean ocean mimetic and ferric iron, and 200% faster in the analogous, ferrous iron containing ocean simulation. Hence, the ferrous condition plausible for the Archean ocean does not only catalyse interconversion among pentose phosphate sugars, they also favoured specificity and increased reactivity in a non-enzymatic, glycolysis-like, chemical reaction system.

The final step in proceedings set up a low concentration of the aforementioned mineral ions, with non-biological optimum temperatures ruling out any enzymes having snuck through, and what&#8217;s more confirming the ferrous iron increases reactivity of glycosysis-like chemical reactions.
What&#8217;s so fascinating about metabolism-first theories is that they still need evolutionary explanation ‒ that is, how did life come to harness these processes, and what did it bring to the table in doing so? Here, the authors note that the evolutionary origins of the Embden-Meyerhof network structure are ‘still largely unknown’.

Geological records reveal details about the chemical environment under which life spread for the first time. This event has been dated between the earliest Archean eon that followed the late heavy bombardment, likely between 4.1 and 3.5 billion years ago, when the first unequivocal traces of life have been dated. Geochemical and geological evidence, particularly the large iron isotope fractionations and the lack of sulphur isotope fractionation during pyrite burial, suggests that the oceans of this period were rich in ferrous iron and poor in sulfate. The absolute concentration of iron in these ancient oceans is equivocal with estimates ranging from 20 lM to 5 mM. Knowledge about the concentrations of other bio-essential metals in the Archean oceans during the evolution of life comes from reconstructing the biogeochemical cycles of these elements and taking into account the environmental properties of a world devoid of oxygen. This has led to the suggestion that copper concentrations in the Archean oceans were negligible and that cobalt and manganese were more abundant than today (but still many orders of magnitude lower than the paleo iron concentrations). The same argument has been applied to zinc; however, its concentration in Proterozoic black shales and Archean iron oxides suggests that its concentration may have remained closer to modern levels over much of the geological time. Nickel and molybdenum concentrations were likely lower than today, whereas concentrations of cobalt and manganese are assumed to be similar to modern levels. It has further been suggested that phosphorus is more easily released from sediment in anoxic (or dysoxic) bottom waters, hinting that the Archean oceans may have been rich in phosphate.

The only flaw in this paper is that in parts it feels a little vague ‒ I don&#8217;t think I&#8217;ll be the only person reading this wondering exactly what the significance of comparing network topology is, but it could well be my own miseducation showing through. Similarly, the how of the origin of life is sparse on detail, but in fairness this is asking a bit much of one paper.

One of the difficulties in describing the origin of metabolism is the fact that the metabolic network is largely composed of intermediates that are not characterized by long-time stability, at least when considering geological environments and timescales. As shown here and previously, this in particular applies to sugar phosphate molecules. In addition, large sugar phosphates are not frequently generated in experiments that address scenarios of primordial carbon fixation. This difficulty cannot, however, mask the fact that sugar phosphates are constituents of many molecules, such as RNA, DNA, ATP and lipids, which are inevitably connected with the emergence of life. It is the fundamental role of sugar phosphates, and the virtual universality of their few metabolic interconversion sequences, that places their origin to the very early evolutionary stages.
Long-term stability seems thus not to be a predictor of whether a molecule adopts a cellular metabolic function. A possible explanation is that molecules stable in a certain environment do not react with their surrounding molecules; inert molecules were thus unlikely to form reaction systems based on environmental catalysts. This seeming paradox of the universality of sugar phosphates and their low stability in the prebiotic world might be solved by accepting different reaction sequences for carbon fixation and the first forms of metabolism: Carbon fixation could have occurred through non-metabolism-like events, including the so-called formose reaction, which is, in a series of condensation steps, able to convert several formaldehyde molecules into complex carbohydrates structures, or through alternative/parallel scenarios that include mineral- or photochemically catalysed reactions, as well as microcompartmentalization, that allowed the accumulation of first biomolecules in protocells. It is only when the first biomolecules had achieved life-compatible concentrations that biologically relevant interconversion sequences, or early forms of metabolic pathways, could become active.

This seems to place their previous results in a bit of a grey area, given that they were produced with the intention of simulating environmental conditions, but in discussing their ideas on the matter the authors are pointing elsewhere for the first sparks of metabolism. The team are the first to point out what they missed — mineral surfaces can act as catalysts; reaction conditions were not optimised to maximise the number of reactions (rather, the reconstruction ran the other way around).

It is thus equally well possible that this part of the pathway came into being with the emergence of enzymes. Therefore, these results could support a hybrid hypothesis to describe the origin of metabolism: a core set of reactions would have be constrained by the environment of the early world organisms, and this network was then extended in terms of both reactivity and efficiency through the evolutionary selection of enzymes until the modern network was in place.

The array of possibilities is staggering. Lastly, it wouldn&#8217;t be an origin of life story without some weird Creature From The Deep, and before finishing the group slip in mention of thermophilic microorganisms that have a functional ‘non-phosphorylating’ Entner-Doudoroff pathway, not yielding any ATP from glucose, just metabolic intermediates. This gives some hope to the possibility of non-ATP-generating glycolysis as described having likewise been a part of the grand scheme of things back in some long-bygone era.

The results presented here demonstrate that sugar phosphate interconversions reactions are prebiotically plausible; thus, the origin of the ribose 5-phosphate through a sugar phosphate interconversion route should be considered. In this context, we have noticed that ribose 5-phosphate was around five times more stable than the other pentose phosphate intermediates (xylulose 5-phosphate and ribulose 5-phosphate) and the formation of ribose 5-phosphate from 6-phosphogluconate was the fastest of all reactions in the presence of Fe(II) (Fig 5A). These properties could have contributed to the central importance of ribose 5-phosphate as backbone of the genetic material.

Looking ahead, the authors propose a deeper inspection of the thermodynamics here, which I look forward to seeing and will fit in nicely with others in the field working towards the same goal.

Studies about the thermodynamics in metabolic systems have, however, shown that reaction equilibrium is shifted under conditions that separate a product from the catalyst, when reactions are coupled and when one product is stabilized, that is, by the inclusion in protocell-like vesicles (Amend et al, 2013). In future studies, it should thus be investigated whether the reactions reported here can be combined with prebiotically plausible mechanisms to stabilize the higher energetic phosphates and thus whether the reactions can be exploited in carbon fixation mechanisms as well.

Update: Larry has now written about this paper himself, and is discussing how he feels it failed to raise contradictions with alternative theories in the comments with co-author Markus Ralser. Interestingly he classifies this as the ‘metabolic soup’ line of thinking, since gluconeogenesis would have sprung up before glycolysis and pentose phosphate pathways.
In particular he seems to feel the authors failed to cite other research (I mean even I knew Nick Lane is one of the eminent scholars in this area yet his work is only cited once, in passing). Food for thought, and a reminder to consider what might be going unsaid. I&#8217;ll have to check back after Ralser has replied, could be an interesting discussion.
⇅ Keller MA, Turchyn AV and Ralser M (2014) Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in the plausible Archean ocean. Molecular Systems Biology 10:725
Above: non-enzymatic sugar phosphate interconversions in a plausible Archean ocean environment reproduce reactions from glycolysis and the pentose phosphate pathways – ‘central carbon metabolism’.
Origins of life on the ocean wave
I don&#8217;t think I&#8217;ve covered theories on biogenesis here before, but for anyone uninitiated on the topic, Larry Moran gave a good summary of the essentials over on his blog in 2009.

There are several competing hypotheses about the origin of life. Most people know about the Primordial Soup scenario; that&#8217;s the one where complex organic molecules are created by spontaneous chemical reactions. Over time these complex molecules, such as amino acids and nucleotides, accumulate in a warm little pond and eventually they come together to form proteins and nucleic acids.
 The RNA World scenario is similar except that nucleic acids (RNA) are thought to form before proteins. For a while, RNA molecules are the main catalysts in the primordial soup. Later on, proteins take over some of the catalytic roles. One of the problems with the RNA world hypothesis is that you have to have a reasonable concentration of nucleotides before the process can begin.
 The third hypothesis is called Metabolism First. In this scheme, the first reactions involve spontaneous formation of simple molecules such as acetate, a two-carbon compound formed from carbon dioxide and water. Pathways leading to the synthesis of simple organic molecules might be promoted by natural catalysts such as minerals and porous surfaces in rocks. The point is that the origin of life is triggered by the accumulation of very simple organic molecules in thermodynamically favorable circumstances.
 Simple organic molecules can then be combined in various ways that result in simple amino acids, lipids, etc. These, in turn, could act as catalysts for the formation of more organic molecules. This is the beginning of metabolism. 
 Eventually simple peptides will be formed and this could lead to better catalysts. Nucleic acids and complex amino acids will only form near the end of this process.

As Larry saw it back then, metabolism-first was the front-runner, offering a simple solution to the problem of life&#8217;s (homo)chirality. Other papers I&#8217;ve seen recently, and the work of Nick Lane (in press at PLOS) who gave a talk at my university last month likewise are in favour metabolism-first.
A new paper from authors split between Cambridge and Mill Hill (London) further bolsters the prospect of metabolism kicking off without enzymes, mimicking the chemical make-up of life&#8217;s earliest oceans in the lab. They found spontaneous occurrence of reaction sequences observed in modern organisms in the glycolysis and the pentose phosphate pathways ‒ ‘central carbon metabolism’.
What&#8217;s more, when iron was maintained ferrous Fe(II), the simulated chemistry of the anoxic Archean ocean stabilised the phosphorylated intermediates and accelerated intermediate reactions and production of pyruvate — a molecule at the intersection of multiple metabolic pathways.

The 29 consequently observed reactions (part c in the main figure above) include the formation of glucose, pyruvate, the nucleic acid precursor ribose-5-phosphate and the amino acid precursor erythrose-4-phosphate. Ferrous iron, Fe(II), is understood to have had high concentrations in the Archean oceans.

The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron-rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.

This is a really nice piece of work, building on earlier pieces that have found the core metabolic network is similar in all organisms. In 2000, Jeong and colleagues — including a couple of names I recognise - Albert and Barabasi — came to this conclusion in a paper on the large-scale organisation of metabolic networks on which I&#8217;ll be posting soon, and just last year Brakman and Smith wrote up a 62 page essay on The Logic of Metabolism which really was quite striking; one of those texts you can&#8217;t put down (seems such a long time ago now!).
» Phys.org: Was life inevitable? New paper pieces together metabolism&#8217;s beginnings» Open access arXiv preprint of the work: q-bio/1207.5532
All this just shows the authors know their field however, and from points made by others they build their investigation: are modern biochemical reaction sequences result of evolutionary selection, or was the initial metabolic reaction network a ready-made entity when life popped up?
As much as the tone is towards the latter, the authors concede both possibilities have their individual merit. Firm experimental evidence for either is sorely lacking.
Fundamental experiments
In water, the group observed 17 cases of glycolytic/pentose phosphate pathway (PPP) intermediates being converted into other metabolites.

In the pentose phosphate pathway, we observed isomerization of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, as well as the formation of glyceraldehyde 3-phosphate from these intermediates (Fig. A, left panel). In cellular metabolism, analogous reaction sequences do exist and are catalysed by the pentose phosphate pathway enzymes ribulose 5-phosphate epimerase, ribose 5-phosphate isomerase and transketolase (Fig. D, left panel). Glycolytic intermediates converted into pyruvate and glucose, the stable products of glycolysis and gluconeogenesis, as well as the intermediate metabolite glucose 6-phosphate (Fig. A, right panel).
Overall, pyruvate formation dominated. Its non-enzymatic formation was detected from PPP metabolites, fructose 6-phosphate, fructose 1,6-bisphosphate and all intermediates of lower glycolysis (Fig. D, right panel). It is therefore apparent that heat exposure is sufficient to convert intermediate metabolites of glycolysis and the pentose phosphate pathway into pyruvate and glucose that constitute thermodynamically stable products also in the modern, enzyme-catalysed metabolism, and to induce isomerization between pentose phosphate metabolites.

To clarify in case it may not be obvious having removed the figure caption, these diagrams show metabolic pathways, or ‘network topologies’ in plain old (high-purity ULC-MS grade) water (A), ‘plausible concentrations’ [for the Archean ocean] of Na, Cl, K, BO3, F, PO4, Mg, Ca, Si, Mo, Co, Ni and Fe (B), a ferrous Fe(II)-supplemented version of this Archean mimetic, i.e. maintaining anoxic conditions (C) and lastly the ‘modern’ enzyme-bolstered glycolysis (canonical Embden-Meyerhof) and pentose phosphate pathways.
After looking at water, the group tested whether conditions replicating the pre-oxygenation oceanic environment would influence these reactions, and as shown above they did, significantly. Amazingly (perhaps, though it does sound labour-intensive) they quantified the metabolites in the resultant 1,200 samples by manually supervised LC-MS/MS peak identification and integration of 18,000 resultant chromatographic peaks. Oceanic salts did not show any significant influence on the sugar phosphate interconversions, which is a notable result in itself.

However, a reactive solution containing the metal ions Fe, Co, Ni, Mo as well as phosphate at Archean ocean plausible concentrations catalysed additional reactions. In the metal-rich solution, out of the 182 reactions monitored, we observed in 28 cases the conversion of a glycolytic or pentose phosphate metabolite into another intermediate of the two pathways (Fig. B). The reactions catalysed by the Archean ocean mimetic yielded the formation of ribose 5-phosphate, the constituent of the RNA backbone and erythrose 4-phosphate, the precursor of aromatic amino acids, starting from several pentose phosphate pathway intermediates.
As a result, these non-enzymatic reactions interconnected all pentose phosphate pathway intermediates (glucose 6-phosphate, erythrose 4-phosphate, ribose 5-phosphate, ribulose 5-phosphate, xylulose 5-phosphate and sedoheptulose 7-phosphate). The interconversion network corresponds to the reaction sequence of the modern-cell non-oxidative pentose pathway (Fig. B and D). In difference, interconversion reactions that would correspond to reactions of the oxidative pentose phosphate pathway, converting glucose 6-phosphate to 6-phosphogluconate, were not observed.

This ocean-in-a-test-tube allowed some neat little tests to be carried out with 6-phosphogluconate (6PG), stable at 70°C in water, but reacting to form PPP intermediates (⇒ pyruvate). The sheer scale of these experiments is impressive ‒ they monitored 182 possible interconversion reactions in the iron-rich mimetic. How many trained lab monkeys does it take to watch 182 reactions you might ask — actually the team used LC-SRM, the second half of which stands for Selected Reaction Monitoring; another form of mass spectrometry. It saw some use in these experiments since it&#8217;s really sensitive (it can measure PTMs below the range of regular mass spec.) and its ‘data-independent nature’.
So as to give more than just a ‘network topology’, they were also able to show that ferrous iron increased specificity, leading to a greater mean carbon recovery in products (62% vs. 50%), i.e. ¼ of the absolute concentrations of non-enzymatically interconverted metabolites formed an intermediate of glycolysis/PPP (calculated with 62% being around ~125% of 50% in case you were wondering!) — i.e. there was quite a favourable yield in this ‘one-pot’ organic synthesis.

The determined reaction rates ranged over four orders of magnitude. In the ferrous iron-rich Archean ocean mimetic, the fastest observed reaction occurred at a rate 19.6 lM/h and was the conversion 6-phosphogluconate to the RNA backbone precursor ribose 5-phosphate. In contrast, the slowest reaction, the dephosphorylation of glucose 6-phosphate to glucose, occurred at a rate of 0.09 lM/h. Several of the interconversion rates were faster in the Archean ocean mimetics compared to the water (12 out of the 17 reactions detected in water); comparing the mimetics, 12 reactions were accelerated in the presence of Fe(II) over Fe(III). 6-phosphogluconate for instance was not converted to ribose 5-phosphate in water. In the ferric iron-rich mimetic, this reaction was observed at a rate of 5.5 lM/h, while in the ferrous iron-rich anoxic experiment at a rate of 19.6 lM/h. In some cases, acceleration was also observed for the three-carbon phosphate interconversion reactions that are analogous to reactions of lower glycolysis; that is, the conversion of phosphoenolpyruvate to pyruvate occurred at 13.5 lM/h in the Fe(II)-rich Archean ocean mimetic, while a rate of 7.3 lM/h was observed in water. However, several of the of three-carbon metabolite formation reactions, including the formation of pyruvate from fructose 1,6-bisphosphate, or glyceraldehyde 3-phosphate from ribose 5-phosphate, were slowed down, most likely due to the stabilization of precursors.
In order to get a global picture of the reactivity in the system, we therefore combined all intermediate metabolites at a concentration of 7.5 lM and determined the temperature-dependent rate of pyruvate formation. At temperatures of 40°C and below, no pyruvate was formed, confirming the absence of enzymatic contamination in the combined reaction mimetic. Above 50°C, pyruvate formation was detected and increased in a temperature-dependent manner. The fastest pyruvate formation rate was detected at 90°C, where typical metabolic enzymes are not functional. Comparing the three conditions, pyruvate formation rate was lowest in water, increased in the presence of the ocean components and achieved its highest rate in the ferrous iron-rich Archean ocean simulation. Compared to water at a temperature of 70°C, the pyruvate formation was 49% faster in the presence of the Archean ocean mimetic and ferric iron, and 200% faster in the analogous, ferrous iron containing ocean simulation. Hence, the ferrous condition plausible for the Archean ocean does not only catalyse interconversion among pentose phosphate sugars, they also favoured specificity and increased reactivity in a non-enzymatic, glycolysis-like, chemical reaction system.

The final step in proceedings set up a low concentration of the aforementioned mineral ions, with non-biological optimum temperatures ruling out any enzymes having snuck through, and what&#8217;s more confirming the ferrous iron increases reactivity of glycosysis-like chemical reactions.
What&#8217;s so fascinating about metabolism-first theories is that they still need evolutionary explanation ‒ that is, how did life come to harness these processes, and what did it bring to the table in doing so? Here, the authors note that the evolutionary origins of the Embden-Meyerhof network structure are ‘still largely unknown’.

Geological records reveal details about the chemical environment under which life spread for the first time. This event has been dated between the earliest Archean eon that followed the late heavy bombardment, likely between 4.1 and 3.5 billion years ago, when the first unequivocal traces of life have been dated. Geochemical and geological evidence, particularly the large iron isotope fractionations and the lack of sulphur isotope fractionation during pyrite burial, suggests that the oceans of this period were rich in ferrous iron and poor in sulfate. The absolute concentration of iron in these ancient oceans is equivocal with estimates ranging from 20 lM to 5 mM. Knowledge about the concentrations of other bio-essential metals in the Archean oceans during the evolution of life comes from reconstructing the biogeochemical cycles of these elements and taking into account the environmental properties of a world devoid of oxygen. This has led to the suggestion that copper concentrations in the Archean oceans were negligible and that cobalt and manganese were more abundant than today (but still many orders of magnitude lower than the paleo iron concentrations). The same argument has been applied to zinc; however, its concentration in Proterozoic black shales and Archean iron oxides suggests that its concentration may have remained closer to modern levels over much of the geological time. Nickel and molybdenum concentrations were likely lower than today, whereas concentrations of cobalt and manganese are assumed to be similar to modern levels. It has further been suggested that phosphorus is more easily released from sediment in anoxic (or dysoxic) bottom waters, hinting that the Archean oceans may have been rich in phosphate.

The only flaw in this paper is that in parts it feels a little vague ‒ I don&#8217;t think I&#8217;ll be the only person reading this wondering exactly what the significance of comparing network topology is, but it could well be my own miseducation showing through. Similarly, the how of the origin of life is sparse on detail, but in fairness this is asking a bit much of one paper.

One of the difficulties in describing the origin of metabolism is the fact that the metabolic network is largely composed of intermediates that are not characterized by long-time stability, at least when considering geological environments and timescales. As shown here and previously, this in particular applies to sugar phosphate molecules. In addition, large sugar phosphates are not frequently generated in experiments that address scenarios of primordial carbon fixation. This difficulty cannot, however, mask the fact that sugar phosphates are constituents of many molecules, such as RNA, DNA, ATP and lipids, which are inevitably connected with the emergence of life. It is the fundamental role of sugar phosphates, and the virtual universality of their few metabolic interconversion sequences, that places their origin to the very early evolutionary stages.
Long-term stability seems thus not to be a predictor of whether a molecule adopts a cellular metabolic function. A possible explanation is that molecules stable in a certain environment do not react with their surrounding molecules; inert molecules were thus unlikely to form reaction systems based on environmental catalysts. This seeming paradox of the universality of sugar phosphates and their low stability in the prebiotic world might be solved by accepting different reaction sequences for carbon fixation and the first forms of metabolism: Carbon fixation could have occurred through non-metabolism-like events, including the so-called formose reaction, which is, in a series of condensation steps, able to convert several formaldehyde molecules into complex carbohydrates structures, or through alternative/parallel scenarios that include mineral- or photochemically catalysed reactions, as well as microcompartmentalization, that allowed the accumulation of first biomolecules in protocells. It is only when the first biomolecules had achieved life-compatible concentrations that biologically relevant interconversion sequences, or early forms of metabolic pathways, could become active.

This seems to place their previous results in a bit of a grey area, given that they were produced with the intention of simulating environmental conditions, but in discussing their ideas on the matter the authors are pointing elsewhere for the first sparks of metabolism. The team are the first to point out what they missed — mineral surfaces can act as catalysts; reaction conditions were not optimised to maximise the number of reactions (rather, the reconstruction ran the other way around).

It is thus equally well possible that this part of the pathway came into being with the emergence of enzymes. Therefore, these results could support a hybrid hypothesis to describe the origin of metabolism: a core set of reactions would have be constrained by the environment of the early world organisms, and this network was then extended in terms of both reactivity and efficiency through the evolutionary selection of enzymes until the modern network was in place.

The array of possibilities is staggering. Lastly, it wouldn&#8217;t be an origin of life story without some weird Creature From The Deep, and before finishing the group slip in mention of thermophilic microorganisms that have a functional ‘non-phosphorylating’ Entner-Doudoroff pathway, not yielding any ATP from glucose, just metabolic intermediates. This gives some hope to the possibility of non-ATP-generating glycolysis as described having likewise been a part of the grand scheme of things back in some long-bygone era.

The results presented here demonstrate that sugar phosphate interconversions reactions are prebiotically plausible; thus, the origin of the ribose 5-phosphate through a sugar phosphate interconversion route should be considered. In this context, we have noticed that ribose 5-phosphate was around five times more stable than the other pentose phosphate intermediates (xylulose 5-phosphate and ribulose 5-phosphate) and the formation of ribose 5-phosphate from 6-phosphogluconate was the fastest of all reactions in the presence of Fe(II) (Fig 5A). These properties could have contributed to the central importance of ribose 5-phosphate as backbone of the genetic material.

Looking ahead, the authors propose a deeper inspection of the thermodynamics here, which I look forward to seeing and will fit in nicely with others in the field working towards the same goal.

Studies about the thermodynamics in metabolic systems have, however, shown that reaction equilibrium is shifted under conditions that separate a product from the catalyst, when reactions are coupled and when one product is stabilized, that is, by the inclusion in protocell-like vesicles (Amend et al, 2013). In future studies, it should thus be investigated whether the reactions reported here can be combined with prebiotically plausible mechanisms to stabilize the higher energetic phosphates and thus whether the reactions can be exploited in carbon fixation mechanisms as well.

Update: Larry has now written about this paper himself, and is discussing how he feels it failed to raise contradictions with alternative theories in the comments with co-author Markus Ralser. Interestingly he classifies this as the ‘metabolic soup’ line of thinking, since gluconeogenesis would have sprung up before glycolysis and pentose phosphate pathways.
In particular he seems to feel the authors failed to cite other research (I mean even I knew Nick Lane is one of the eminent scholars in this area yet his work is only cited once, in passing). Food for thought, and a reminder to consider what might be going unsaid. I&#8217;ll have to check back after Ralser has replied, could be an interesting discussion.
⇅ Keller MA, Turchyn AV and Ralser M (2014) Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in the plausible Archean ocean. Molecular Systems Biology 10:725
Above: non-enzymatic sugar phosphate interconversions in a plausible Archean ocean environment reproduce reactions from glycolysis and the pentose phosphate pathways – ‘central carbon metabolism’.
Origins of life on the ocean wave
I don&#8217;t think I&#8217;ve covered theories on biogenesis here before, but for anyone uninitiated on the topic, Larry Moran gave a good summary of the essentials over on his blog in 2009.

There are several competing hypotheses about the origin of life. Most people know about the Primordial Soup scenario; that&#8217;s the one where complex organic molecules are created by spontaneous chemical reactions. Over time these complex molecules, such as amino acids and nucleotides, accumulate in a warm little pond and eventually they come together to form proteins and nucleic acids.
 The RNA World scenario is similar except that nucleic acids (RNA) are thought to form before proteins. For a while, RNA molecules are the main catalysts in the primordial soup. Later on, proteins take over some of the catalytic roles. One of the problems with the RNA world hypothesis is that you have to have a reasonable concentration of nucleotides before the process can begin.
 The third hypothesis is called Metabolism First. In this scheme, the first reactions involve spontaneous formation of simple molecules such as acetate, a two-carbon compound formed from carbon dioxide and water. Pathways leading to the synthesis of simple organic molecules might be promoted by natural catalysts such as minerals and porous surfaces in rocks. The point is that the origin of life is triggered by the accumulation of very simple organic molecules in thermodynamically favorable circumstances.
 Simple organic molecules can then be combined in various ways that result in simple amino acids, lipids, etc. These, in turn, could act as catalysts for the formation of more organic molecules. This is the beginning of metabolism. 
 Eventually simple peptides will be formed and this could lead to better catalysts. Nucleic acids and complex amino acids will only form near the end of this process.

As Larry saw it back then, metabolism-first was the front-runner, offering a simple solution to the problem of life&#8217;s (homo)chirality. Other papers I&#8217;ve seen recently, and the work of Nick Lane (in press at PLOS) who gave a talk at my university last month likewise are in favour metabolism-first.
A new paper from authors split between Cambridge and Mill Hill (London) further bolsters the prospect of metabolism kicking off without enzymes, mimicking the chemical make-up of life&#8217;s earliest oceans in the lab. They found spontaneous occurrence of reaction sequences observed in modern organisms in the glycolysis and the pentose phosphate pathways ‒ ‘central carbon metabolism’.
What&#8217;s more, when iron was maintained ferrous Fe(II), the simulated chemistry of the anoxic Archean ocean stabilised the phosphorylated intermediates and accelerated intermediate reactions and production of pyruvate — a molecule at the intersection of multiple metabolic pathways.

The 29 consequently observed reactions (part c in the main figure above) include the formation of glucose, pyruvate, the nucleic acid precursor ribose-5-phosphate and the amino acid precursor erythrose-4-phosphate. Ferrous iron, Fe(II), is understood to have had high concentrations in the Archean oceans.

The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron-rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.

This is a really nice piece of work, building on earlier pieces that have found the core metabolic network is similar in all organisms. In 2000, Jeong and colleagues — including a couple of names I recognise - Albert and Barabasi — came to this conclusion in a paper on the large-scale organisation of metabolic networks on which I&#8217;ll be posting soon, and just last year Brakman and Smith wrote up a 62 page essay on The Logic of Metabolism which really was quite striking; one of those texts you can&#8217;t put down (seems such a long time ago now!).
» Phys.org: Was life inevitable? New paper pieces together metabolism&#8217;s beginnings» Open access arXiv preprint of the work: q-bio/1207.5532
All this just shows the authors know their field however, and from points made by others they build their investigation: are modern biochemical reaction sequences result of evolutionary selection, or was the initial metabolic reaction network a ready-made entity when life popped up?
As much as the tone is towards the latter, the authors concede both possibilities have their individual merit. Firm experimental evidence for either is sorely lacking.
Fundamental experiments
In water, the group observed 17 cases of glycolytic/pentose phosphate pathway (PPP) intermediates being converted into other metabolites.

In the pentose phosphate pathway, we observed isomerization of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, as well as the formation of glyceraldehyde 3-phosphate from these intermediates (Fig. A, left panel). In cellular metabolism, analogous reaction sequences do exist and are catalysed by the pentose phosphate pathway enzymes ribulose 5-phosphate epimerase, ribose 5-phosphate isomerase and transketolase (Fig. D, left panel). Glycolytic intermediates converted into pyruvate and glucose, the stable products of glycolysis and gluconeogenesis, as well as the intermediate metabolite glucose 6-phosphate (Fig. A, right panel).
Overall, pyruvate formation dominated. Its non-enzymatic formation was detected from PPP metabolites, fructose 6-phosphate, fructose 1,6-bisphosphate and all intermediates of lower glycolysis (Fig. D, right panel). It is therefore apparent that heat exposure is sufficient to convert intermediate metabolites of glycolysis and the pentose phosphate pathway into pyruvate and glucose that constitute thermodynamically stable products also in the modern, enzyme-catalysed metabolism, and to induce isomerization between pentose phosphate metabolites.

To clarify in case it may not be obvious having removed the figure caption, these diagrams show metabolic pathways, or ‘network topologies’ in plain old (high-purity ULC-MS grade) water (A), ‘plausible concentrations’ [for the Archean ocean] of Na, Cl, K, BO3, F, PO4, Mg, Ca, Si, Mo, Co, Ni and Fe (B), a ferrous Fe(II)-supplemented version of this Archean mimetic, i.e. maintaining anoxic conditions (C) and lastly the ‘modern’ enzyme-bolstered glycolysis (canonical Embden-Meyerhof) and pentose phosphate pathways.
After looking at water, the group tested whether conditions replicating the pre-oxygenation oceanic environment would influence these reactions, and as shown above they did, significantly. Amazingly (perhaps, though it does sound labour-intensive) they quantified the metabolites in the resultant 1,200 samples by manually supervised LC-MS/MS peak identification and integration of 18,000 resultant chromatographic peaks. Oceanic salts did not show any significant influence on the sugar phosphate interconversions, which is a notable result in itself.

However, a reactive solution containing the metal ions Fe, Co, Ni, Mo as well as phosphate at Archean ocean plausible concentrations catalysed additional reactions. In the metal-rich solution, out of the 182 reactions monitored, we observed in 28 cases the conversion of a glycolytic or pentose phosphate metabolite into another intermediate of the two pathways (Fig. B). The reactions catalysed by the Archean ocean mimetic yielded the formation of ribose 5-phosphate, the constituent of the RNA backbone and erythrose 4-phosphate, the precursor of aromatic amino acids, starting from several pentose phosphate pathway intermediates.
As a result, these non-enzymatic reactions interconnected all pentose phosphate pathway intermediates (glucose 6-phosphate, erythrose 4-phosphate, ribose 5-phosphate, ribulose 5-phosphate, xylulose 5-phosphate and sedoheptulose 7-phosphate). The interconversion network corresponds to the reaction sequence of the modern-cell non-oxidative pentose pathway (Fig. B and D). In difference, interconversion reactions that would correspond to reactions of the oxidative pentose phosphate pathway, converting glucose 6-phosphate to 6-phosphogluconate, were not observed.

This ocean-in-a-test-tube allowed some neat little tests to be carried out with 6-phosphogluconate (6PG), stable at 70°C in water, but reacting to form PPP intermediates (⇒ pyruvate). The sheer scale of these experiments is impressive ‒ they monitored 182 possible interconversion reactions in the iron-rich mimetic. How many trained lab monkeys does it take to watch 182 reactions you might ask — actually the team used LC-SRM, the second half of which stands for Selected Reaction Monitoring; another form of mass spectrometry. It saw some use in these experiments since it&#8217;s really sensitive (it can measure PTMs below the range of regular mass spec.) and its ‘data-independent nature’.
So as to give more than just a ‘network topology’, they were also able to show that ferrous iron increased specificity, leading to a greater mean carbon recovery in products (62% vs. 50%), i.e. ¼ of the absolute concentrations of non-enzymatically interconverted metabolites formed an intermediate of glycolysis/PPP (calculated with 62% being around ~125% of 50% in case you were wondering!) — i.e. there was quite a favourable yield in this ‘one-pot’ organic synthesis.

The determined reaction rates ranged over four orders of magnitude. In the ferrous iron-rich Archean ocean mimetic, the fastest observed reaction occurred at a rate 19.6 lM/h and was the conversion 6-phosphogluconate to the RNA backbone precursor ribose 5-phosphate. In contrast, the slowest reaction, the dephosphorylation of glucose 6-phosphate to glucose, occurred at a rate of 0.09 lM/h. Several of the interconversion rates were faster in the Archean ocean mimetics compared to the water (12 out of the 17 reactions detected in water); comparing the mimetics, 12 reactions were accelerated in the presence of Fe(II) over Fe(III). 6-phosphogluconate for instance was not converted to ribose 5-phosphate in water. In the ferric iron-rich mimetic, this reaction was observed at a rate of 5.5 lM/h, while in the ferrous iron-rich anoxic experiment at a rate of 19.6 lM/h. In some cases, acceleration was also observed for the three-carbon phosphate interconversion reactions that are analogous to reactions of lower glycolysis; that is, the conversion of phosphoenolpyruvate to pyruvate occurred at 13.5 lM/h in the Fe(II)-rich Archean ocean mimetic, while a rate of 7.3 lM/h was observed in water. However, several of the of three-carbon metabolite formation reactions, including the formation of pyruvate from fructose 1,6-bisphosphate, or glyceraldehyde 3-phosphate from ribose 5-phosphate, were slowed down, most likely due to the stabilization of precursors.
In order to get a global picture of the reactivity in the system, we therefore combined all intermediate metabolites at a concentration of 7.5 lM and determined the temperature-dependent rate of pyruvate formation. At temperatures of 40°C and below, no pyruvate was formed, confirming the absence of enzymatic contamination in the combined reaction mimetic. Above 50°C, pyruvate formation was detected and increased in a temperature-dependent manner. The fastest pyruvate formation rate was detected at 90°C, where typical metabolic enzymes are not functional. Comparing the three conditions, pyruvate formation rate was lowest in water, increased in the presence of the ocean components and achieved its highest rate in the ferrous iron-rich Archean ocean simulation. Compared to water at a temperature of 70°C, the pyruvate formation was 49% faster in the presence of the Archean ocean mimetic and ferric iron, and 200% faster in the analogous, ferrous iron containing ocean simulation. Hence, the ferrous condition plausible for the Archean ocean does not only catalyse interconversion among pentose phosphate sugars, they also favoured specificity and increased reactivity in a non-enzymatic, glycolysis-like, chemical reaction system.

The final step in proceedings set up a low concentration of the aforementioned mineral ions, with non-biological optimum temperatures ruling out any enzymes having snuck through, and what&#8217;s more confirming the ferrous iron increases reactivity of glycosysis-like chemical reactions.
What&#8217;s so fascinating about metabolism-first theories is that they still need evolutionary explanation ‒ that is, how did life come to harness these processes, and what did it bring to the table in doing so? Here, the authors note that the evolutionary origins of the Embden-Meyerhof network structure are ‘still largely unknown’.

Geological records reveal details about the chemical environment under which life spread for the first time. This event has been dated between the earliest Archean eon that followed the late heavy bombardment, likely between 4.1 and 3.5 billion years ago, when the first unequivocal traces of life have been dated. Geochemical and geological evidence, particularly the large iron isotope fractionations and the lack of sulphur isotope fractionation during pyrite burial, suggests that the oceans of this period were rich in ferrous iron and poor in sulfate. The absolute concentration of iron in these ancient oceans is equivocal with estimates ranging from 20 lM to 5 mM. Knowledge about the concentrations of other bio-essential metals in the Archean oceans during the evolution of life comes from reconstructing the biogeochemical cycles of these elements and taking into account the environmental properties of a world devoid of oxygen. This has led to the suggestion that copper concentrations in the Archean oceans were negligible and that cobalt and manganese were more abundant than today (but still many orders of magnitude lower than the paleo iron concentrations). The same argument has been applied to zinc; however, its concentration in Proterozoic black shales and Archean iron oxides suggests that its concentration may have remained closer to modern levels over much of the geological time. Nickel and molybdenum concentrations were likely lower than today, whereas concentrations of cobalt and manganese are assumed to be similar to modern levels. It has further been suggested that phosphorus is more easily released from sediment in anoxic (or dysoxic) bottom waters, hinting that the Archean oceans may have been rich in phosphate.

The only flaw in this paper is that in parts it feels a little vague ‒ I don&#8217;t think I&#8217;ll be the only person reading this wondering exactly what the significance of comparing network topology is, but it could well be my own miseducation showing through. Similarly, the how of the origin of life is sparse on detail, but in fairness this is asking a bit much of one paper.

One of the difficulties in describing the origin of metabolism is the fact that the metabolic network is largely composed of intermediates that are not characterized by long-time stability, at least when considering geological environments and timescales. As shown here and previously, this in particular applies to sugar phosphate molecules. In addition, large sugar phosphates are not frequently generated in experiments that address scenarios of primordial carbon fixation. This difficulty cannot, however, mask the fact that sugar phosphates are constituents of many molecules, such as RNA, DNA, ATP and lipids, which are inevitably connected with the emergence of life. It is the fundamental role of sugar phosphates, and the virtual universality of their few metabolic interconversion sequences, that places their origin to the very early evolutionary stages.
Long-term stability seems thus not to be a predictor of whether a molecule adopts a cellular metabolic function. A possible explanation is that molecules stable in a certain environment do not react with their surrounding molecules; inert molecules were thus unlikely to form reaction systems based on environmental catalysts. This seeming paradox of the universality of sugar phosphates and their low stability in the prebiotic world might be solved by accepting different reaction sequences for carbon fixation and the first forms of metabolism: Carbon fixation could have occurred through non-metabolism-like events, including the so-called formose reaction, which is, in a series of condensation steps, able to convert several formaldehyde molecules into complex carbohydrates structures, or through alternative/parallel scenarios that include mineral- or photochemically catalysed reactions, as well as microcompartmentalization, that allowed the accumulation of first biomolecules in protocells. It is only when the first biomolecules had achieved life-compatible concentrations that biologically relevant interconversion sequences, or early forms of metabolic pathways, could become active.

This seems to place their previous results in a bit of a grey area, given that they were produced with the intention of simulating environmental conditions, but in discussing their ideas on the matter the authors are pointing elsewhere for the first sparks of metabolism. The team are the first to point out what they missed — mineral surfaces can act as catalysts; reaction conditions were not optimised to maximise the number of reactions (rather, the reconstruction ran the other way around).

It is thus equally well possible that this part of the pathway came into being with the emergence of enzymes. Therefore, these results could support a hybrid hypothesis to describe the origin of metabolism: a core set of reactions would have be constrained by the environment of the early world organisms, and this network was then extended in terms of both reactivity and efficiency through the evolutionary selection of enzymes until the modern network was in place.

The array of possibilities is staggering. Lastly, it wouldn&#8217;t be an origin of life story without some weird Creature From The Deep, and before finishing the group slip in mention of thermophilic microorganisms that have a functional ‘non-phosphorylating’ Entner-Doudoroff pathway, not yielding any ATP from glucose, just metabolic intermediates. This gives some hope to the possibility of non-ATP-generating glycolysis as described having likewise been a part of the grand scheme of things back in some long-bygone era.

The results presented here demonstrate that sugar phosphate interconversions reactions are prebiotically plausible; thus, the origin of the ribose 5-phosphate through a sugar phosphate interconversion route should be considered. In this context, we have noticed that ribose 5-phosphate was around five times more stable than the other pentose phosphate intermediates (xylulose 5-phosphate and ribulose 5-phosphate) and the formation of ribose 5-phosphate from 6-phosphogluconate was the fastest of all reactions in the presence of Fe(II) (Fig 5A). These properties could have contributed to the central importance of ribose 5-phosphate as backbone of the genetic material.

Looking ahead, the authors propose a deeper inspection of the thermodynamics here, which I look forward to seeing and will fit in nicely with others in the field working towards the same goal.

Studies about the thermodynamics in metabolic systems have, however, shown that reaction equilibrium is shifted under conditions that separate a product from the catalyst, when reactions are coupled and when one product is stabilized, that is, by the inclusion in protocell-like vesicles (Amend et al, 2013). In future studies, it should thus be investigated whether the reactions reported here can be combined with prebiotically plausible mechanisms to stabilize the higher energetic phosphates and thus whether the reactions can be exploited in carbon fixation mechanisms as well.

Update: Larry has now written about this paper himself, and is discussing how he feels it failed to raise contradictions with alternative theories in the comments with co-author Markus Ralser. Interestingly he classifies this as the ‘metabolic soup’ line of thinking, since gluconeogenesis would have sprung up before glycolysis and pentose phosphate pathways.
In particular he seems to feel the authors failed to cite other research (I mean even I knew Nick Lane is one of the eminent scholars in this area yet his work is only cited once, in passing). Food for thought, and a reminder to consider what might be going unsaid. I&#8217;ll have to check back after Ralser has replied, could be an interesting discussion.
⇅ Keller MA, Turchyn AV and Ralser M (2014) Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in the plausible Archean ocean. Molecular Systems Biology 10:725
Above: non-enzymatic sugar phosphate interconversions in a plausible Archean ocean environment reproduce reactions from glycolysis and the pentose phosphate pathways – ‘central carbon metabolism’.
Origins of life on the ocean wave
I don&#8217;t think I&#8217;ve covered theories on biogenesis here before, but for anyone uninitiated on the topic, Larry Moran gave a good summary of the essentials over on his blog in 2009.

There are several competing hypotheses about the origin of life. Most people know about the Primordial Soup scenario; that&#8217;s the one where complex organic molecules are created by spontaneous chemical reactions. Over time these complex molecules, such as amino acids and nucleotides, accumulate in a warm little pond and eventually they come together to form proteins and nucleic acids.
 The RNA World scenario is similar except that nucleic acids (RNA) are thought to form before proteins. For a while, RNA molecules are the main catalysts in the primordial soup. Later on, proteins take over some of the catalytic roles. One of the problems with the RNA world hypothesis is that you have to have a reasonable concentration of nucleotides before the process can begin.
 The third hypothesis is called Metabolism First. In this scheme, the first reactions involve spontaneous formation of simple molecules such as acetate, a two-carbon compound formed from carbon dioxide and water. Pathways leading to the synthesis of simple organic molecules might be promoted by natural catalysts such as minerals and porous surfaces in rocks. The point is that the origin of life is triggered by the accumulation of very simple organic molecules in thermodynamically favorable circumstances.
 Simple organic molecules can then be combined in various ways that result in simple amino acids, lipids, etc. These, in turn, could act as catalysts for the formation of more organic molecules. This is the beginning of metabolism. 
 Eventually simple peptides will be formed and this could lead to better catalysts. Nucleic acids and complex amino acids will only form near the end of this process.

As Larry saw it back then, metabolism-first was the front-runner, offering a simple solution to the problem of life&#8217;s (homo)chirality. Other papers I&#8217;ve seen recently, and the work of Nick Lane (in press at PLOS) who gave a talk at my university last month likewise are in favour metabolism-first.
A new paper from authors split between Cambridge and Mill Hill (London) further bolsters the prospect of metabolism kicking off without enzymes, mimicking the chemical make-up of life&#8217;s earliest oceans in the lab. They found spontaneous occurrence of reaction sequences observed in modern organisms in the glycolysis and the pentose phosphate pathways ‒ ‘central carbon metabolism’.
What&#8217;s more, when iron was maintained ferrous Fe(II), the simulated chemistry of the anoxic Archean ocean stabilised the phosphorylated intermediates and accelerated intermediate reactions and production of pyruvate — a molecule at the intersection of multiple metabolic pathways.

The 29 consequently observed reactions (part c in the main figure above) include the formation of glucose, pyruvate, the nucleic acid precursor ribose-5-phosphate and the amino acid precursor erythrose-4-phosphate. Ferrous iron, Fe(II), is understood to have had high concentrations in the Archean oceans.

The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron-rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.

This is a really nice piece of work, building on earlier pieces that have found the core metabolic network is similar in all organisms. In 2000, Jeong and colleagues — including a couple of names I recognise - Albert and Barabasi — came to this conclusion in a paper on the large-scale organisation of metabolic networks on which I&#8217;ll be posting soon, and just last year Brakman and Smith wrote up a 62 page essay on The Logic of Metabolism which really was quite striking; one of those texts you can&#8217;t put down (seems such a long time ago now!).
» Phys.org: Was life inevitable? New paper pieces together metabolism&#8217;s beginnings» Open access arXiv preprint of the work: q-bio/1207.5532
All this just shows the authors know their field however, and from points made by others they build their investigation: are modern biochemical reaction sequences result of evolutionary selection, or was the initial metabolic reaction network a ready-made entity when life popped up?
As much as the tone is towards the latter, the authors concede both possibilities have their individual merit. Firm experimental evidence for either is sorely lacking.
Fundamental experiments
In water, the group observed 17 cases of glycolytic/pentose phosphate pathway (PPP) intermediates being converted into other metabolites.

In the pentose phosphate pathway, we observed isomerization of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, as well as the formation of glyceraldehyde 3-phosphate from these intermediates (Fig. A, left panel). In cellular metabolism, analogous reaction sequences do exist and are catalysed by the pentose phosphate pathway enzymes ribulose 5-phosphate epimerase, ribose 5-phosphate isomerase and transketolase (Fig. D, left panel). Glycolytic intermediates converted into pyruvate and glucose, the stable products of glycolysis and gluconeogenesis, as well as the intermediate metabolite glucose 6-phosphate (Fig. A, right panel).
Overall, pyruvate formation dominated. Its non-enzymatic formation was detected from PPP metabolites, fructose 6-phosphate, fructose 1,6-bisphosphate and all intermediates of lower glycolysis (Fig. D, right panel). It is therefore apparent that heat exposure is sufficient to convert intermediate metabolites of glycolysis and the pentose phosphate pathway into pyruvate and glucose that constitute thermodynamically stable products also in the modern, enzyme-catalysed metabolism, and to induce isomerization between pentose phosphate metabolites.

To clarify in case it may not be obvious having removed the figure caption, these diagrams show metabolic pathways, or ‘network topologies’ in plain old (high-purity ULC-MS grade) water (A), ‘plausible concentrations’ [for the Archean ocean] of Na, Cl, K, BO3, F, PO4, Mg, Ca, Si, Mo, Co, Ni and Fe (B), a ferrous Fe(II)-supplemented version of this Archean mimetic, i.e. maintaining anoxic conditions (C) and lastly the ‘modern’ enzyme-bolstered glycolysis (canonical Embden-Meyerhof) and pentose phosphate pathways.
After looking at water, the group tested whether conditions replicating the pre-oxygenation oceanic environment would influence these reactions, and as shown above they did, significantly. Amazingly (perhaps, though it does sound labour-intensive) they quantified the metabolites in the resultant 1,200 samples by manually supervised LC-MS/MS peak identification and integration of 18,000 resultant chromatographic peaks. Oceanic salts did not show any significant influence on the sugar phosphate interconversions, which is a notable result in itself.

However, a reactive solution containing the metal ions Fe, Co, Ni, Mo as well as phosphate at Archean ocean plausible concentrations catalysed additional reactions. In the metal-rich solution, out of the 182 reactions monitored, we observed in 28 cases the conversion of a glycolytic or pentose phosphate metabolite into another intermediate of the two pathways (Fig. B). The reactions catalysed by the Archean ocean mimetic yielded the formation of ribose 5-phosphate, the constituent of the RNA backbone and erythrose 4-phosphate, the precursor of aromatic amino acids, starting from several pentose phosphate pathway intermediates.
As a result, these non-enzymatic reactions interconnected all pentose phosphate pathway intermediates (glucose 6-phosphate, erythrose 4-phosphate, ribose 5-phosphate, ribulose 5-phosphate, xylulose 5-phosphate and sedoheptulose 7-phosphate). The interconversion network corresponds to the reaction sequence of the modern-cell non-oxidative pentose pathway (Fig. B and D). In difference, interconversion reactions that would correspond to reactions of the oxidative pentose phosphate pathway, converting glucose 6-phosphate to 6-phosphogluconate, were not observed.

This ocean-in-a-test-tube allowed some neat little tests to be carried out with 6-phosphogluconate (6PG), stable at 70°C in water, but reacting to form PPP intermediates (⇒ pyruvate). The sheer scale of these experiments is impressive ‒ they monitored 182 possible interconversion reactions in the iron-rich mimetic. How many trained lab monkeys does it take to watch 182 reactions you might ask — actually the team used LC-SRM, the second half of which stands for Selected Reaction Monitoring; another form of mass spectrometry. It saw some use in these experiments since it&#8217;s really sensitive (it can measure PTMs below the range of regular mass spec.) and its ‘data-independent nature’.
So as to give more than just a ‘network topology’, they were also able to show that ferrous iron increased specificity, leading to a greater mean carbon recovery in products (62% vs. 50%), i.e. ¼ of the absolute concentrations of non-enzymatically interconverted metabolites formed an intermediate of glycolysis/PPP (calculated with 62% being around ~125% of 50% in case you were wondering!) — i.e. there was quite a favourable yield in this ‘one-pot’ organic synthesis.

The determined reaction rates ranged over four orders of magnitude. In the ferrous iron-rich Archean ocean mimetic, the fastest observed reaction occurred at a rate 19.6 lM/h and was the conversion 6-phosphogluconate to the RNA backbone precursor ribose 5-phosphate. In contrast, the slowest reaction, the dephosphorylation of glucose 6-phosphate to glucose, occurred at a rate of 0.09 lM/h. Several of the interconversion rates were faster in the Archean ocean mimetics compared to the water (12 out of the 17 reactions detected in water); comparing the mimetics, 12 reactions were accelerated in the presence of Fe(II) over Fe(III). 6-phosphogluconate for instance was not converted to ribose 5-phosphate in water. In the ferric iron-rich mimetic, this reaction was observed at a rate of 5.5 lM/h, while in the ferrous iron-rich anoxic experiment at a rate of 19.6 lM/h. In some cases, acceleration was also observed for the three-carbon phosphate interconversion reactions that are analogous to reactions of lower glycolysis; that is, the conversion of phosphoenolpyruvate to pyruvate occurred at 13.5 lM/h in the Fe(II)-rich Archean ocean mimetic, while a rate of 7.3 lM/h was observed in water. However, several of the of three-carbon metabolite formation reactions, including the formation of pyruvate from fructose 1,6-bisphosphate, or glyceraldehyde 3-phosphate from ribose 5-phosphate, were slowed down, most likely due to the stabilization of precursors.
In order to get a global picture of the reactivity in the system, we therefore combined all intermediate metabolites at a concentration of 7.5 lM and determined the temperature-dependent rate of pyruvate formation. At temperatures of 40°C and below, no pyruvate was formed, confirming the absence of enzymatic contamination in the combined reaction mimetic. Above 50°C, pyruvate formation was detected and increased in a temperature-dependent manner. The fastest pyruvate formation rate was detected at 90°C, where typical metabolic enzymes are not functional. Comparing the three conditions, pyruvate formation rate was lowest in water, increased in the presence of the ocean components and achieved its highest rate in the ferrous iron-rich Archean ocean simulation. Compared to water at a temperature of 70°C, the pyruvate formation was 49% faster in the presence of the Archean ocean mimetic and ferric iron, and 200% faster in the analogous, ferrous iron containing ocean simulation. Hence, the ferrous condition plausible for the Archean ocean does not only catalyse interconversion among pentose phosphate sugars, they also favoured specificity and increased reactivity in a non-enzymatic, glycolysis-like, chemical reaction system.

The final step in proceedings set up a low concentration of the aforementioned mineral ions, with non-biological optimum temperatures ruling out any enzymes having snuck through, and what&#8217;s more confirming the ferrous iron increases reactivity of glycosysis-like chemical reactions.
What&#8217;s so fascinating about metabolism-first theories is that they still need evolutionary explanation ‒ that is, how did life come to harness these processes, and what did it bring to the table in doing so? Here, the authors note that the evolutionary origins of the Embden-Meyerhof network structure are ‘still largely unknown’.

Geological records reveal details about the chemical environment under which life spread for the first time. This event has been dated between the earliest Archean eon that followed the late heavy bombardment, likely between 4.1 and 3.5 billion years ago, when the first unequivocal traces of life have been dated. Geochemical and geological evidence, particularly the large iron isotope fractionations and the lack of sulphur isotope fractionation during pyrite burial, suggests that the oceans of this period were rich in ferrous iron and poor in sulfate. The absolute concentration of iron in these ancient oceans is equivocal with estimates ranging from 20 lM to 5 mM. Knowledge about the concentrations of other bio-essential metals in the Archean oceans during the evolution of life comes from reconstructing the biogeochemical cycles of these elements and taking into account the environmental properties of a world devoid of oxygen. This has led to the suggestion that copper concentrations in the Archean oceans were negligible and that cobalt and manganese were more abundant than today (but still many orders of magnitude lower than the paleo iron concentrations). The same argument has been applied to zinc; however, its concentration in Proterozoic black shales and Archean iron oxides suggests that its concentration may have remained closer to modern levels over much of the geological time. Nickel and molybdenum concentrations were likely lower than today, whereas concentrations of cobalt and manganese are assumed to be similar to modern levels. It has further been suggested that phosphorus is more easily released from sediment in anoxic (or dysoxic) bottom waters, hinting that the Archean oceans may have been rich in phosphate.

The only flaw in this paper is that in parts it feels a little vague ‒ I don&#8217;t think I&#8217;ll be the only person reading this wondering exactly what the significance of comparing network topology is, but it could well be my own miseducation showing through. Similarly, the how of the origin of life is sparse on detail, but in fairness this is asking a bit much of one paper.

One of the difficulties in describing the origin of metabolism is the fact that the metabolic network is largely composed of intermediates that are not characterized by long-time stability, at least when considering geological environments and timescales. As shown here and previously, this in particular applies to sugar phosphate molecules. In addition, large sugar phosphates are not frequently generated in experiments that address scenarios of primordial carbon fixation. This difficulty cannot, however, mask the fact that sugar phosphates are constituents of many molecules, such as RNA, DNA, ATP and lipids, which are inevitably connected with the emergence of life. It is the fundamental role of sugar phosphates, and the virtual universality of their few metabolic interconversion sequences, that places their origin to the very early evolutionary stages.
Long-term stability seems thus not to be a predictor of whether a molecule adopts a cellular metabolic function. A possible explanation is that molecules stable in a certain environment do not react with their surrounding molecules; inert molecules were thus unlikely to form reaction systems based on environmental catalysts. This seeming paradox of the universality of sugar phosphates and their low stability in the prebiotic world might be solved by accepting different reaction sequences for carbon fixation and the first forms of metabolism: Carbon fixation could have occurred through non-metabolism-like events, including the so-called formose reaction, which is, in a series of condensation steps, able to convert several formaldehyde molecules into complex carbohydrates structures, or through alternative/parallel scenarios that include mineral- or photochemically catalysed reactions, as well as microcompartmentalization, that allowed the accumulation of first biomolecules in protocells. It is only when the first biomolecules had achieved life-compatible concentrations that biologically relevant interconversion sequences, or early forms of metabolic pathways, could become active.

This seems to place their previous results in a bit of a grey area, given that they were produced with the intention of simulating environmental conditions, but in discussing their ideas on the matter the authors are pointing elsewhere for the first sparks of metabolism. The team are the first to point out what they missed — mineral surfaces can act as catalysts; reaction conditions were not optimised to maximise the number of reactions (rather, the reconstruction ran the other way around).

It is thus equally well possible that this part of the pathway came into being with the emergence of enzymes. Therefore, these results could support a hybrid hypothesis to describe the origin of metabolism: a core set of reactions would have be constrained by the environment of the early world organisms, and this network was then extended in terms of both reactivity and efficiency through the evolutionary selection of enzymes until the modern network was in place.

The array of possibilities is staggering. Lastly, it wouldn&#8217;t be an origin of life story without some weird Creature From The Deep, and before finishing the group slip in mention of thermophilic microorganisms that have a functional ‘non-phosphorylating’ Entner-Doudoroff pathway, not yielding any ATP from glucose, just metabolic intermediates. This gives some hope to the possibility of non-ATP-generating glycolysis as described having likewise been a part of the grand scheme of things back in some long-bygone era.

The results presented here demonstrate that sugar phosphate interconversions reactions are prebiotically plausible; thus, the origin of the ribose 5-phosphate through a sugar phosphate interconversion route should be considered. In this context, we have noticed that ribose 5-phosphate was around five times more stable than the other pentose phosphate intermediates (xylulose 5-phosphate and ribulose 5-phosphate) and the formation of ribose 5-phosphate from 6-phosphogluconate was the fastest of all reactions in the presence of Fe(II) (Fig 5A). These properties could have contributed to the central importance of ribose 5-phosphate as backbone of the genetic material.

Looking ahead, the authors propose a deeper inspection of the thermodynamics here, which I look forward to seeing and will fit in nicely with others in the field working towards the same goal.

Studies about the thermodynamics in metabolic systems have, however, shown that reaction equilibrium is shifted under conditions that separate a product from the catalyst, when reactions are coupled and when one product is stabilized, that is, by the inclusion in protocell-like vesicles (Amend et al, 2013). In future studies, it should thus be investigated whether the reactions reported here can be combined with prebiotically plausible mechanisms to stabilize the higher energetic phosphates and thus whether the reactions can be exploited in carbon fixation mechanisms as well.

Update: Larry has now written about this paper himself, and is discussing how he feels it failed to raise contradictions with alternative theories in the comments with co-author Markus Ralser. Interestingly he classifies this as the ‘metabolic soup’ line of thinking, since gluconeogenesis would have sprung up before glycolysis and pentose phosphate pathways.
In particular he seems to feel the authors failed to cite other research (I mean even I knew Nick Lane is one of the eminent scholars in this area yet his work is only cited once, in passing). Food for thought, and a reminder to consider what might be going unsaid. I&#8217;ll have to check back after Ralser has replied, could be an interesting discussion.
⇅ Keller MA, Turchyn AV and Ralser M (2014) Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in the plausible Archean ocean. Molecular Systems Biology 10:725
Above: non-enzymatic sugar phosphate interconversions in a plausible Archean ocean environment reproduce reactions from glycolysis and the pentose phosphate pathways – ‘central carbon metabolism’.
Origins of life on the ocean wave
I don&#8217;t think I&#8217;ve covered theories on biogenesis here before, but for anyone uninitiated on the topic, Larry Moran gave a good summary of the essentials over on his blog in 2009.

There are several competing hypotheses about the origin of life. Most people know about the Primordial Soup scenario; that&#8217;s the one where complex organic molecules are created by spontaneous chemical reactions. Over time these complex molecules, such as amino acids and nucleotides, accumulate in a warm little pond and eventually they come together to form proteins and nucleic acids.
 The RNA World scenario is similar except that nucleic acids (RNA) are thought to form before proteins. For a while, RNA molecules are the main catalysts in the primordial soup. Later on, proteins take over some of the catalytic roles. One of the problems with the RNA world hypothesis is that you have to have a reasonable concentration of nucleotides before the process can begin.
 The third hypothesis is called Metabolism First. In this scheme, the first reactions involve spontaneous formation of simple molecules such as acetate, a two-carbon compound formed from carbon dioxide and water. Pathways leading to the synthesis of simple organic molecules might be promoted by natural catalysts such as minerals and porous surfaces in rocks. The point is that the origin of life is triggered by the accumulation of very simple organic molecules in thermodynamically favorable circumstances.
 Simple organic molecules can then be combined in various ways that result in simple amino acids, lipids, etc. These, in turn, could act as catalysts for the formation of more organic molecules. This is the beginning of metabolism. 
 Eventually simple peptides will be formed and this could lead to better catalysts. Nucleic acids and complex amino acids will only form near the end of this process.

As Larry saw it back then, metabolism-first was the front-runner, offering a simple solution to the problem of life&#8217;s (homo)chirality. Other papers I&#8217;ve seen recently, and the work of Nick Lane (in press at PLOS) who gave a talk at my university last month likewise are in favour metabolism-first.
A new paper from authors split between Cambridge and Mill Hill (London) further bolsters the prospect of metabolism kicking off without enzymes, mimicking the chemical make-up of life&#8217;s earliest oceans in the lab. They found spontaneous occurrence of reaction sequences observed in modern organisms in the glycolysis and the pentose phosphate pathways ‒ ‘central carbon metabolism’.
What&#8217;s more, when iron was maintained ferrous Fe(II), the simulated chemistry of the anoxic Archean ocean stabilised the phosphorylated intermediates and accelerated intermediate reactions and production of pyruvate — a molecule at the intersection of multiple metabolic pathways.

The 29 consequently observed reactions (part c in the main figure above) include the formation of glucose, pyruvate, the nucleic acid precursor ribose-5-phosphate and the amino acid precursor erythrose-4-phosphate. Ferrous iron, Fe(II), is understood to have had high concentrations in the Archean oceans.

The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron-rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.

This is a really nice piece of work, building on earlier pieces that have found the core metabolic network is similar in all organisms. In 2000, Jeong and colleagues — including a couple of names I recognise - Albert and Barabasi — came to this conclusion in a paper on the large-scale organisation of metabolic networks on which I&#8217;ll be posting soon, and just last year Brakman and Smith wrote up a 62 page essay on The Logic of Metabolism which really was quite striking; one of those texts you can&#8217;t put down (seems such a long time ago now!).
» Phys.org: Was life inevitable? New paper pieces together metabolism&#8217;s beginnings» Open access arXiv preprint of the work: q-bio/1207.5532
All this just shows the authors know their field however, and from points made by others they build their investigation: are modern biochemical reaction sequences result of evolutionary selection, or was the initial metabolic reaction network a ready-made entity when life popped up?
As much as the tone is towards the latter, the authors concede both possibilities have their individual merit. Firm experimental evidence for either is sorely lacking.
Fundamental experiments
In water, the group observed 17 cases of glycolytic/pentose phosphate pathway (PPP) intermediates being converted into other metabolites.

In the pentose phosphate pathway, we observed isomerization of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, as well as the formation of glyceraldehyde 3-phosphate from these intermediates (Fig. A, left panel). In cellular metabolism, analogous reaction sequences do exist and are catalysed by the pentose phosphate pathway enzymes ribulose 5-phosphate epimerase, ribose 5-phosphate isomerase and transketolase (Fig. D, left panel). Glycolytic intermediates converted into pyruvate and glucose, the stable products of glycolysis and gluconeogenesis, as well as the intermediate metabolite glucose 6-phosphate (Fig. A, right panel).
Overall, pyruvate formation dominated. Its non-enzymatic formation was detected from PPP metabolites, fructose 6-phosphate, fructose 1,6-bisphosphate and all intermediates of lower glycolysis (Fig. D, right panel). It is therefore apparent that heat exposure is sufficient to convert intermediate metabolites of glycolysis and the pentose phosphate pathway into pyruvate and glucose that constitute thermodynamically stable products also in the modern, enzyme-catalysed metabolism, and to induce isomerization between pentose phosphate metabolites.

To clarify in case it may not be obvious having removed the figure caption, these diagrams show metabolic pathways, or ‘network topologies’ in plain old (high-purity ULC-MS grade) water (A), ‘plausible concentrations’ [for the Archean ocean] of Na, Cl, K, BO3, F, PO4, Mg, Ca, Si, Mo, Co, Ni and Fe (B), a ferrous Fe(II)-supplemented version of this Archean mimetic, i.e. maintaining anoxic conditions (C) and lastly the ‘modern’ enzyme-bolstered glycolysis (canonical Embden-Meyerhof) and pentose phosphate pathways.
After looking at water, the group tested whether conditions replicating the pre-oxygenation oceanic environment would influence these reactions, and as shown above they did, significantly. Amazingly (perhaps, though it does sound labour-intensive) they quantified the metabolites in the resultant 1,200 samples by manually supervised LC-MS/MS peak identification and integration of 18,000 resultant chromatographic peaks. Oceanic salts did not show any significant influence on the sugar phosphate interconversions, which is a notable result in itself.

However, a reactive solution containing the metal ions Fe, Co, Ni, Mo as well as phosphate at Archean ocean plausible concentrations catalysed additional reactions. In the metal-rich solution, out of the 182 reactions monitored, we observed in 28 cases the conversion of a glycolytic or pentose phosphate metabolite into another intermediate of the two pathways (Fig. B). The reactions catalysed by the Archean ocean mimetic yielded the formation of ribose 5-phosphate, the constituent of the RNA backbone and erythrose 4-phosphate, the precursor of aromatic amino acids, starting from several pentose phosphate pathway intermediates.
As a result, these non-enzymatic reactions interconnected all pentose phosphate pathway intermediates (glucose 6-phosphate, erythrose 4-phosphate, ribose 5-phosphate, ribulose 5-phosphate, xylulose 5-phosphate and sedoheptulose 7-phosphate). The interconversion network corresponds to the reaction sequence of the modern-cell non-oxidative pentose pathway (Fig. B and D). In difference, interconversion reactions that would correspond to reactions of the oxidative pentose phosphate pathway, converting glucose 6-phosphate to 6-phosphogluconate, were not observed.

This ocean-in-a-test-tube allowed some neat little tests to be carried out with 6-phosphogluconate (6PG), stable at 70°C in water, but reacting to form PPP intermediates (⇒ pyruvate). The sheer scale of these experiments is impressive ‒ they monitored 182 possible interconversion reactions in the iron-rich mimetic. How many trained lab monkeys does it take to watch 182 reactions you might ask — actually the team used LC-SRM, the second half of which stands for Selected Reaction Monitoring; another form of mass spectrometry. It saw some use in these experiments since it&#8217;s really sensitive (it can measure PTMs below the range of regular mass spec.) and its ‘data-independent nature’.
So as to give more than just a ‘network topology’, they were also able to show that ferrous iron increased specificity, leading to a greater mean carbon recovery in products (62% vs. 50%), i.e. ¼ of the absolute concentrations of non-enzymatically interconverted metabolites formed an intermediate of glycolysis/PPP (calculated with 62% being around ~125% of 50% in case you were wondering!) — i.e. there was quite a favourable yield in this ‘one-pot’ organic synthesis.

The determined reaction rates ranged over four orders of magnitude. In the ferrous iron-rich Archean ocean mimetic, the fastest observed reaction occurred at a rate 19.6 lM/h and was the conversion 6-phosphogluconate to the RNA backbone precursor ribose 5-phosphate. In contrast, the slowest reaction, the dephosphorylation of glucose 6-phosphate to glucose, occurred at a rate of 0.09 lM/h. Several of the interconversion rates were faster in the Archean ocean mimetics compared to the water (12 out of the 17 reactions detected in water); comparing the mimetics, 12 reactions were accelerated in the presence of Fe(II) over Fe(III). 6-phosphogluconate for instance was not converted to ribose 5-phosphate in water. In the ferric iron-rich mimetic, this reaction was observed at a rate of 5.5 lM/h, while in the ferrous iron-rich anoxic experiment at a rate of 19.6 lM/h. In some cases, acceleration was also observed for the three-carbon phosphate interconversion reactions that are analogous to reactions of lower glycolysis; that is, the conversion of phosphoenolpyruvate to pyruvate occurred at 13.5 lM/h in the Fe(II)-rich Archean ocean mimetic, while a rate of 7.3 lM/h was observed in water. However, several of the of three-carbon metabolite formation reactions, including the formation of pyruvate from fructose 1,6-bisphosphate, or glyceraldehyde 3-phosphate from ribose 5-phosphate, were slowed down, most likely due to the stabilization of precursors.
In order to get a global picture of the reactivity in the system, we therefore combined all intermediate metabolites at a concentration of 7.5 lM and determined the temperature-dependent rate of pyruvate formation. At temperatures of 40°C and below, no pyruvate was formed, confirming the absence of enzymatic contamination in the combined reaction mimetic. Above 50°C, pyruvate formation was detected and increased in a temperature-dependent manner. The fastest pyruvate formation rate was detected at 90°C, where typical metabolic enzymes are not functional. Comparing the three conditions, pyruvate formation rate was lowest in water, increased in the presence of the ocean components and achieved its highest rate in the ferrous iron-rich Archean ocean simulation. Compared to water at a temperature of 70°C, the pyruvate formation was 49% faster in the presence of the Archean ocean mimetic and ferric iron, and 200% faster in the analogous, ferrous iron containing ocean simulation. Hence, the ferrous condition plausible for the Archean ocean does not only catalyse interconversion among pentose phosphate sugars, they also favoured specificity and increased reactivity in a non-enzymatic, glycolysis-like, chemical reaction system.

The final step in proceedings set up a low concentration of the aforementioned mineral ions, with non-biological optimum temperatures ruling out any enzymes having snuck through, and what&#8217;s more confirming the ferrous iron increases reactivity of glycosysis-like chemical reactions.
What&#8217;s so fascinating about metabolism-first theories is that they still need evolutionary explanation ‒ that is, how did life come to harness these processes, and what did it bring to the table in doing so? Here, the authors note that the evolutionary origins of the Embden-Meyerhof network structure are ‘still largely unknown’.

Geological records reveal details about the chemical environment under which life spread for the first time. This event has been dated between the earliest Archean eon that followed the late heavy bombardment, likely between 4.1 and 3.5 billion years ago, when the first unequivocal traces of life have been dated. Geochemical and geological evidence, particularly the large iron isotope fractionations and the lack of sulphur isotope fractionation during pyrite burial, suggests that the oceans of this period were rich in ferrous iron and poor in sulfate. The absolute concentration of iron in these ancient oceans is equivocal with estimates ranging from 20 lM to 5 mM. Knowledge about the concentrations of other bio-essential metals in the Archean oceans during the evolution of life comes from reconstructing the biogeochemical cycles of these elements and taking into account the environmental properties of a world devoid of oxygen. This has led to the suggestion that copper concentrations in the Archean oceans were negligible and that cobalt and manganese were more abundant than today (but still many orders of magnitude lower than the paleo iron concentrations). The same argument has been applied to zinc; however, its concentration in Proterozoic black shales and Archean iron oxides suggests that its concentration may have remained closer to modern levels over much of the geological time. Nickel and molybdenum concentrations were likely lower than today, whereas concentrations of cobalt and manganese are assumed to be similar to modern levels. It has further been suggested that phosphorus is more easily released from sediment in anoxic (or dysoxic) bottom waters, hinting that the Archean oceans may have been rich in phosphate.

The only flaw in this paper is that in parts it feels a little vague ‒ I don&#8217;t think I&#8217;ll be the only person reading this wondering exactly what the significance of comparing network topology is, but it could well be my own miseducation showing through. Similarly, the how of the origin of life is sparse on detail, but in fairness this is asking a bit much of one paper.

One of the difficulties in describing the origin of metabolism is the fact that the metabolic network is largely composed of intermediates that are not characterized by long-time stability, at least when considering geological environments and timescales. As shown here and previously, this in particular applies to sugar phosphate molecules. In addition, large sugar phosphates are not frequently generated in experiments that address scenarios of primordial carbon fixation. This difficulty cannot, however, mask the fact that sugar phosphates are constituents of many molecules, such as RNA, DNA, ATP and lipids, which are inevitably connected with the emergence of life. It is the fundamental role of sugar phosphates, and the virtual universality of their few metabolic interconversion sequences, that places their origin to the very early evolutionary stages.
Long-term stability seems thus not to be a predictor of whether a molecule adopts a cellular metabolic function. A possible explanation is that molecules stable in a certain environment do not react with their surrounding molecules; inert molecules were thus unlikely to form reaction systems based on environmental catalysts. This seeming paradox of the universality of sugar phosphates and their low stability in the prebiotic world might be solved by accepting different reaction sequences for carbon fixation and the first forms of metabolism: Carbon fixation could have occurred through non-metabolism-like events, including the so-called formose reaction, which is, in a series of condensation steps, able to convert several formaldehyde molecules into complex carbohydrates structures, or through alternative/parallel scenarios that include mineral- or photochemically catalysed reactions, as well as microcompartmentalization, that allowed the accumulation of first biomolecules in protocells. It is only when the first biomolecules had achieved life-compatible concentrations that biologically relevant interconversion sequences, or early forms of metabolic pathways, could become active.

This seems to place their previous results in a bit of a grey area, given that they were produced with the intention of simulating environmental conditions, but in discussing their ideas on the matter the authors are pointing elsewhere for the first sparks of metabolism. The team are the first to point out what they missed — mineral surfaces can act as catalysts; reaction conditions were not optimised to maximise the number of reactions (rather, the reconstruction ran the other way around).

It is thus equally well possible that this part of the pathway came into being with the emergence of enzymes. Therefore, these results could support a hybrid hypothesis to describe the origin of metabolism: a core set of reactions would have be constrained by the environment of the early world organisms, and this network was then extended in terms of both reactivity and efficiency through the evolutionary selection of enzymes until the modern network was in place.

The array of possibilities is staggering. Lastly, it wouldn&#8217;t be an origin of life story without some weird Creature From The Deep, and before finishing the group slip in mention of thermophilic microorganisms that have a functional ‘non-phosphorylating’ Entner-Doudoroff pathway, not yielding any ATP from glucose, just metabolic intermediates. This gives some hope to the possibility of non-ATP-generating glycolysis as described having likewise been a part of the grand scheme of things back in some long-bygone era.

The results presented here demonstrate that sugar phosphate interconversions reactions are prebiotically plausible; thus, the origin of the ribose 5-phosphate through a sugar phosphate interconversion route should be considered. In this context, we have noticed that ribose 5-phosphate was around five times more stable than the other pentose phosphate intermediates (xylulose 5-phosphate and ribulose 5-phosphate) and the formation of ribose 5-phosphate from 6-phosphogluconate was the fastest of all reactions in the presence of Fe(II) (Fig 5A). These properties could have contributed to the central importance of ribose 5-phosphate as backbone of the genetic material.

Looking ahead, the authors propose a deeper inspection of the thermodynamics here, which I look forward to seeing and will fit in nicely with others in the field working towards the same goal.

Studies about the thermodynamics in metabolic systems have, however, shown that reaction equilibrium is shifted under conditions that separate a product from the catalyst, when reactions are coupled and when one product is stabilized, that is, by the inclusion in protocell-like vesicles (Amend et al, 2013). In future studies, it should thus be investigated whether the reactions reported here can be combined with prebiotically plausible mechanisms to stabilize the higher energetic phosphates and thus whether the reactions can be exploited in carbon fixation mechanisms as well.

Update: Larry has now written about this paper himself, and is discussing how he feels it failed to raise contradictions with alternative theories in the comments with co-author Markus Ralser. Interestingly he classifies this as the ‘metabolic soup’ line of thinking, since gluconeogenesis would have sprung up before glycolysis and pentose phosphate pathways.
In particular he seems to feel the authors failed to cite other research (I mean even I knew Nick Lane is one of the eminent scholars in this area yet his work is only cited once, in passing). Food for thought, and a reminder to consider what might be going unsaid. I&#8217;ll have to check back after Ralser has replied, could be an interesting discussion.
⇅ Keller MA, Turchyn AV and Ralser M (2014) Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in the plausible Archean ocean. Molecular Systems Biology 10:725
Above: non-enzymatic sugar phosphate interconversions in a plausible Archean ocean environment reproduce reactions from glycolysis and the pentose phosphate pathways – ‘central carbon metabolism’.
Origins of life on the ocean wave
I don&#8217;t think I&#8217;ve covered theories on biogenesis here before, but for anyone uninitiated on the topic, Larry Moran gave a good summary of the essentials over on his blog in 2009.

There are several competing hypotheses about the origin of life. Most people know about the Primordial Soup scenario; that&#8217;s the one where complex organic molecules are created by spontaneous chemical reactions. Over time these complex molecules, such as amino acids and nucleotides, accumulate in a warm little pond and eventually they come together to form proteins and nucleic acids.
 The RNA World scenario is similar except that nucleic acids (RNA) are thought to form before proteins. For a while, RNA molecules are the main catalysts in the primordial soup. Later on, proteins take over some of the catalytic roles. One of the problems with the RNA world hypothesis is that you have to have a reasonable concentration of nucleotides before the process can begin.
 The third hypothesis is called Metabolism First. In this scheme, the first reactions involve spontaneous formation of simple molecules such as acetate, a two-carbon compound formed from carbon dioxide and water. Pathways leading to the synthesis of simple organic molecules might be promoted by natural catalysts such as minerals and porous surfaces in rocks. The point is that the origin of life is triggered by the accumulation of very simple organic molecules in thermodynamically favorable circumstances.
 Simple organic molecules can then be combined in various ways that result in simple amino acids, lipids, etc. These, in turn, could act as catalysts for the formation of more organic molecules. This is the beginning of metabolism. 
 Eventually simple peptides will be formed and this could lead to better catalysts. Nucleic acids and complex amino acids will only form near the end of this process.

As Larry saw it back then, metabolism-first was the front-runner, offering a simple solution to the problem of life&#8217;s (homo)chirality. Other papers I&#8217;ve seen recently, and the work of Nick Lane (in press at PLOS) who gave a talk at my university last month likewise are in favour metabolism-first.
A new paper from authors split between Cambridge and Mill Hill (London) further bolsters the prospect of metabolism kicking off without enzymes, mimicking the chemical make-up of life&#8217;s earliest oceans in the lab. They found spontaneous occurrence of reaction sequences observed in modern organisms in the glycolysis and the pentose phosphate pathways ‒ ‘central carbon metabolism’.
What&#8217;s more, when iron was maintained ferrous Fe(II), the simulated chemistry of the anoxic Archean ocean stabilised the phosphorylated intermediates and accelerated intermediate reactions and production of pyruvate — a molecule at the intersection of multiple metabolic pathways.

The 29 consequently observed reactions (part c in the main figure above) include the formation of glucose, pyruvate, the nucleic acid precursor ribose-5-phosphate and the amino acid precursor erythrose-4-phosphate. Ferrous iron, Fe(II), is understood to have had high concentrations in the Archean oceans.

The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron-rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.

This is a really nice piece of work, building on earlier pieces that have found the core metabolic network is similar in all organisms. In 2000, Jeong and colleagues — including a couple of names I recognise - Albert and Barabasi — came to this conclusion in a paper on the large-scale organisation of metabolic networks on which I&#8217;ll be posting soon, and just last year Brakman and Smith wrote up a 62 page essay on The Logic of Metabolism which really was quite striking; one of those texts you can&#8217;t put down (seems such a long time ago now!).
» Phys.org: Was life inevitable? New paper pieces together metabolism&#8217;s beginnings» Open access arXiv preprint of the work: q-bio/1207.5532
All this just shows the authors know their field however, and from points made by others they build their investigation: are modern biochemical reaction sequences result of evolutionary selection, or was the initial metabolic reaction network a ready-made entity when life popped up?
As much as the tone is towards the latter, the authors concede both possibilities have their individual merit. Firm experimental evidence for either is sorely lacking.
Fundamental experiments
In water, the group observed 17 cases of glycolytic/pentose phosphate pathway (PPP) intermediates being converted into other metabolites.

In the pentose phosphate pathway, we observed isomerization of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, as well as the formation of glyceraldehyde 3-phosphate from these intermediates (Fig. A, left panel). In cellular metabolism, analogous reaction sequences do exist and are catalysed by the pentose phosphate pathway enzymes ribulose 5-phosphate epimerase, ribose 5-phosphate isomerase and transketolase (Fig. D, left panel). Glycolytic intermediates converted into pyruvate and glucose, the stable products of glycolysis and gluconeogenesis, as well as the intermediate metabolite glucose 6-phosphate (Fig. A, right panel).
Overall, pyruvate formation dominated. Its non-enzymatic formation was detected from PPP metabolites, fructose 6-phosphate, fructose 1,6-bisphosphate and all intermediates of lower glycolysis (Fig. D, right panel). It is therefore apparent that heat exposure is sufficient to convert intermediate metabolites of glycolysis and the pentose phosphate pathway into pyruvate and glucose that constitute thermodynamically stable products also in the modern, enzyme-catalysed metabolism, and to induce isomerization between pentose phosphate metabolites.

To clarify in case it may not be obvious having removed the figure caption, these diagrams show metabolic pathways, or ‘network topologies’ in plain old (high-purity ULC-MS grade) water (A), ‘plausible concentrations’ [for the Archean ocean] of Na, Cl, K, BO3, F, PO4, Mg, Ca, Si, Mo, Co, Ni and Fe (B), a ferrous Fe(II)-supplemented version of this Archean mimetic, i.e. maintaining anoxic conditions (C) and lastly the ‘modern’ enzyme-bolstered glycolysis (canonical Embden-Meyerhof) and pentose phosphate pathways.
After looking at water, the group tested whether conditions replicating the pre-oxygenation oceanic environment would influence these reactions, and as shown above they did, significantly. Amazingly (perhaps, though it does sound labour-intensive) they quantified the metabolites in the resultant 1,200 samples by manually supervised LC-MS/MS peak identification and integration of 18,000 resultant chromatographic peaks. Oceanic salts did not show any significant influence on the sugar phosphate interconversions, which is a notable result in itself.

However, a reactive solution containing the metal ions Fe, Co, Ni, Mo as well as phosphate at Archean ocean plausible concentrations catalysed additional reactions. In the metal-rich solution, out of the 182 reactions monitored, we observed in 28 cases the conversion of a glycolytic or pentose phosphate metabolite into another intermediate of the two pathways (Fig. B). The reactions catalysed by the Archean ocean mimetic yielded the formation of ribose 5-phosphate, the constituent of the RNA backbone and erythrose 4-phosphate, the precursor of aromatic amino acids, starting from several pentose phosphate pathway intermediates.
As a result, these non-enzymatic reactions interconnected all pentose phosphate pathway intermediates (glucose 6-phosphate, erythrose 4-phosphate, ribose 5-phosphate, ribulose 5-phosphate, xylulose 5-phosphate and sedoheptulose 7-phosphate). The interconversion network corresponds to the reaction sequence of the modern-cell non-oxidative pentose pathway (Fig. B and D). In difference, interconversion reactions that would correspond to reactions of the oxidative pentose phosphate pathway, converting glucose 6-phosphate to 6-phosphogluconate, were not observed.

This ocean-in-a-test-tube allowed some neat little tests to be carried out with 6-phosphogluconate (6PG), stable at 70°C in water, but reacting to form PPP intermediates (⇒ pyruvate). The sheer scale of these experiments is impressive ‒ they monitored 182 possible interconversion reactions in the iron-rich mimetic. How many trained lab monkeys does it take to watch 182 reactions you might ask — actually the team used LC-SRM, the second half of which stands for Selected Reaction Monitoring; another form of mass spectrometry. It saw some use in these experiments since it&#8217;s really sensitive (it can measure PTMs below the range of regular mass spec.) and its ‘data-independent nature’.
So as to give more than just a ‘network topology’, they were also able to show that ferrous iron increased specificity, leading to a greater mean carbon recovery in products (62% vs. 50%), i.e. ¼ of the absolute concentrations of non-enzymatically interconverted metabolites formed an intermediate of glycolysis/PPP (calculated with 62% being around ~125% of 50% in case you were wondering!) — i.e. there was quite a favourable yield in this ‘one-pot’ organic synthesis.

The determined reaction rates ranged over four orders of magnitude. In the ferrous iron-rich Archean ocean mimetic, the fastest observed reaction occurred at a rate 19.6 lM/h and was the conversion 6-phosphogluconate to the RNA backbone precursor ribose 5-phosphate. In contrast, the slowest reaction, the dephosphorylation of glucose 6-phosphate to glucose, occurred at a rate of 0.09 lM/h. Several of the interconversion rates were faster in the Archean ocean mimetics compared to the water (12 out of the 17 reactions detected in water); comparing the mimetics, 12 reactions were accelerated in the presence of Fe(II) over Fe(III). 6-phosphogluconate for instance was not converted to ribose 5-phosphate in water. In the ferric iron-rich mimetic, this reaction was observed at a rate of 5.5 lM/h, while in the ferrous iron-rich anoxic experiment at a rate of 19.6 lM/h. In some cases, acceleration was also observed for the three-carbon phosphate interconversion reactions that are analogous to reactions of lower glycolysis; that is, the conversion of phosphoenolpyruvate to pyruvate occurred at 13.5 lM/h in the Fe(II)-rich Archean ocean mimetic, while a rate of 7.3 lM/h was observed in water. However, several of the of three-carbon metabolite formation reactions, including the formation of pyruvate from fructose 1,6-bisphosphate, or glyceraldehyde 3-phosphate from ribose 5-phosphate, were slowed down, most likely due to the stabilization of precursors.
In order to get a global picture of the reactivity in the system, we therefore combined all intermediate metabolites at a concentration of 7.5 lM and determined the temperature-dependent rate of pyruvate formation. At temperatures of 40°C and below, no pyruvate was formed, confirming the absence of enzymatic contamination in the combined reaction mimetic. Above 50°C, pyruvate formation was detected and increased in a temperature-dependent manner. The fastest pyruvate formation rate was detected at 90°C, where typical metabolic enzymes are not functional. Comparing the three conditions, pyruvate formation rate was lowest in water, increased in the presence of the ocean components and achieved its highest rate in the ferrous iron-rich Archean ocean simulation. Compared to water at a temperature of 70°C, the pyruvate formation was 49% faster in the presence of the Archean ocean mimetic and ferric iron, and 200% faster in the analogous, ferrous iron containing ocean simulation. Hence, the ferrous condition plausible for the Archean ocean does not only catalyse interconversion among pentose phosphate sugars, they also favoured specificity and increased reactivity in a non-enzymatic, glycolysis-like, chemical reaction system.

The final step in proceedings set up a low concentration of the aforementioned mineral ions, with non-biological optimum temperatures ruling out any enzymes having snuck through, and what&#8217;s more confirming the ferrous iron increases reactivity of glycosysis-like chemical reactions.
What&#8217;s so fascinating about metabolism-first theories is that they still need evolutionary explanation ‒ that is, how did life come to harness these processes, and what did it bring to the table in doing so? Here, the authors note that the evolutionary origins of the Embden-Meyerhof network structure are ‘still largely unknown’.

Geological records reveal details about the chemical environment under which life spread for the first time. This event has been dated between the earliest Archean eon that followed the late heavy bombardment, likely between 4.1 and 3.5 billion years ago, when the first unequivocal traces of life have been dated. Geochemical and geological evidence, particularly the large iron isotope fractionations and the lack of sulphur isotope fractionation during pyrite burial, suggests that the oceans of this period were rich in ferrous iron and poor in sulfate. The absolute concentration of iron in these ancient oceans is equivocal with estimates ranging from 20 lM to 5 mM. Knowledge about the concentrations of other bio-essential metals in the Archean oceans during the evolution of life comes from reconstructing the biogeochemical cycles of these elements and taking into account the environmental properties of a world devoid of oxygen. This has led to the suggestion that copper concentrations in the Archean oceans were negligible and that cobalt and manganese were more abundant than today (but still many orders of magnitude lower than the paleo iron concentrations). The same argument has been applied to zinc; however, its concentration in Proterozoic black shales and Archean iron oxides suggests that its concentration may have remained closer to modern levels over much of the geological time. Nickel and molybdenum concentrations were likely lower than today, whereas concentrations of cobalt and manganese are assumed to be similar to modern levels. It has further been suggested that phosphorus is more easily released from sediment in anoxic (or dysoxic) bottom waters, hinting that the Archean oceans may have been rich in phosphate.

The only flaw in this paper is that in parts it feels a little vague ‒ I don&#8217;t think I&#8217;ll be the only person reading this wondering exactly what the significance of comparing network topology is, but it could well be my own miseducation showing through. Similarly, the how of the origin of life is sparse on detail, but in fairness this is asking a bit much of one paper.

One of the difficulties in describing the origin of metabolism is the fact that the metabolic network is largely composed of intermediates that are not characterized by long-time stability, at least when considering geological environments and timescales. As shown here and previously, this in particular applies to sugar phosphate molecules. In addition, large sugar phosphates are not frequently generated in experiments that address scenarios of primordial carbon fixation. This difficulty cannot, however, mask the fact that sugar phosphates are constituents of many molecules, such as RNA, DNA, ATP and lipids, which are inevitably connected with the emergence of life. It is the fundamental role of sugar phosphates, and the virtual universality of their few metabolic interconversion sequences, that places their origin to the very early evolutionary stages.
Long-term stability seems thus not to be a predictor of whether a molecule adopts a cellular metabolic function. A possible explanation is that molecules stable in a certain environment do not react with their surrounding molecules; inert molecules were thus unlikely to form reaction systems based on environmental catalysts. This seeming paradox of the universality of sugar phosphates and their low stability in the prebiotic world might be solved by accepting different reaction sequences for carbon fixation and the first forms of metabolism: Carbon fixation could have occurred through non-metabolism-like events, including the so-called formose reaction, which is, in a series of condensation steps, able to convert several formaldehyde molecules into complex carbohydrates structures, or through alternative/parallel scenarios that include mineral- or photochemically catalysed reactions, as well as microcompartmentalization, that allowed the accumulation of first biomolecules in protocells. It is only when the first biomolecules had achieved life-compatible concentrations that biologically relevant interconversion sequences, or early forms of metabolic pathways, could become active.

This seems to place their previous results in a bit of a grey area, given that they were produced with the intention of simulating environmental conditions, but in discussing their ideas on the matter the authors are pointing elsewhere for the first sparks of metabolism. The team are the first to point out what they missed — mineral surfaces can act as catalysts; reaction conditions were not optimised to maximise the number of reactions (rather, the reconstruction ran the other way around).

It is thus equally well possible that this part of the pathway came into being with the emergence of enzymes. Therefore, these results could support a hybrid hypothesis to describe the origin of metabolism: a core set of reactions would have be constrained by the environment of the early world organisms, and this network was then extended in terms of both reactivity and efficiency through the evolutionary selection of enzymes until the modern network was in place.

The array of possibilities is staggering. Lastly, it wouldn&#8217;t be an origin of life story without some weird Creature From The Deep, and before finishing the group slip in mention of thermophilic microorganisms that have a functional ‘non-phosphorylating’ Entner-Doudoroff pathway, not yielding any ATP from glucose, just metabolic intermediates. This gives some hope to the possibility of non-ATP-generating glycolysis as described having likewise been a part of the grand scheme of things back in some long-bygone era.

The results presented here demonstrate that sugar phosphate interconversions reactions are prebiotically plausible; thus, the origin of the ribose 5-phosphate through a sugar phosphate interconversion route should be considered. In this context, we have noticed that ribose 5-phosphate was around five times more stable than the other pentose phosphate intermediates (xylulose 5-phosphate and ribulose 5-phosphate) and the formation of ribose 5-phosphate from 6-phosphogluconate was the fastest of all reactions in the presence of Fe(II) (Fig 5A). These properties could have contributed to the central importance of ribose 5-phosphate as backbone of the genetic material.

Looking ahead, the authors propose a deeper inspection of the thermodynamics here, which I look forward to seeing and will fit in nicely with others in the field working towards the same goal.

Studies about the thermodynamics in metabolic systems have, however, shown that reaction equilibrium is shifted under conditions that separate a product from the catalyst, when reactions are coupled and when one product is stabilized, that is, by the inclusion in protocell-like vesicles (Amend et al, 2013). In future studies, it should thus be investigated whether the reactions reported here can be combined with prebiotically plausible mechanisms to stabilize the higher energetic phosphates and thus whether the reactions can be exploited in carbon fixation mechanisms as well.

Update: Larry has now written about this paper himself, and is discussing how he feels it failed to raise contradictions with alternative theories in the comments with co-author Markus Ralser. Interestingly he classifies this as the ‘metabolic soup’ line of thinking, since gluconeogenesis would have sprung up before glycolysis and pentose phosphate pathways.
In particular he seems to feel the authors failed to cite other research (I mean even I knew Nick Lane is one of the eminent scholars in this area yet his work is only cited once, in passing). Food for thought, and a reminder to consider what might be going unsaid. I&#8217;ll have to check back after Ralser has replied, could be an interesting discussion.
⇅ Keller MA, Turchyn AV and Ralser M (2014) Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in the plausible Archean ocean. Molecular Systems Biology 10:725
Above: non-enzymatic sugar phosphate interconversions in a plausible Archean ocean environment reproduce reactions from glycolysis and the pentose phosphate pathways – ‘central carbon metabolism’.

Origins of life on the ocean wave

I don’t think I’ve covered theories on biogenesis here before, but for anyone uninitiated on the topic, Larry Moran gave a good summary of the essentials over on his blog in 2009.

There are several competing hypotheses about the origin of life. Most people know about the Primordial Soup scenario; that’s the one where complex organic molecules are created by spontaneous chemical reactions. Over time these complex molecules, such as amino acids and nucleotides, accumulate in a warm little pond and eventually they come together to form proteins and nucleic acids.

The RNA World scenario is similar except that nucleic acids (RNA) are thought to form before proteins. For a while, RNA molecules are the main catalysts in the primordial soup. Later on, proteins take over some of the catalytic roles. One of the problems with the RNA world hypothesis is that you have to have a reasonable concentration of nucleotides before the process can begin.

The third hypothesis is called Metabolism First. In this scheme, the first reactions involve spontaneous formation of simple molecules such as acetate, a two-carbon compound formed from carbon dioxide and water. Pathways leading to the synthesis of simple organic molecules might be promoted by natural catalysts such as minerals and porous surfaces in rocks. The point is that the origin of life is triggered by the accumulation of very simple organic molecules in thermodynamically favorable circumstances.

Simple organic molecules can then be combined in various ways that result in simple amino acids, lipids, etc. These, in turn, could act as catalysts for the formation of more organic molecules. This is the beginning of metabolism. 

Eventually simple peptides will be formed and this could lead to better catalysts. Nucleic acids and complex amino acids will only form near the end of this process.

As Larry saw it back then, metabolism-first was the front-runner, offering a simple solution to the problem of life’s (homo)chirality. Other papers I’ve seen recently, and the work of Nick Lane (in press at PLOS) who gave a talk at my university last month likewise are in favour metabolism-first.

A new paper from authors split between Cambridge and Mill Hill (London) further bolsters the prospect of metabolism kicking off without enzymes, mimicking the chemical make-up of life’s earliest oceans in the lab. They found spontaneous occurrence of reaction sequences observed in modern organisms in the glycolysis and the pentose phosphate pathways ‒ ‘central carbon metabolism’.

What’s more, when iron was maintained ferrous Fe(II), the simulated chemistry of the anoxic Archean ocean stabilised the phosphorylated intermediates and accelerated intermediate reactions and production of pyruvate — a molecule at the intersection of multiple metabolic pathways.

image

The 29 consequently observed reactions (part c in the main figure above) include the formation of glucose, pyruvate, the nucleic acid precursor ribose-5-phosphate and the amino acid precursor erythrose-4-phosphate. Ferrous iron, Fe(II), is understood to have had high concentrations in the Archean oceans.

The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron-rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.

This is a really nice piece of work, building on earlier pieces that have found the core metabolic network is similar in all organisms. In 2000, Jeong and colleagues — including a couple of names I recognise - Albert and Barabasi — came to this conclusion in a paper on the large-scale organisation of metabolic networks on which I’ll be posting soon, and just last year Brakman and Smith wrote up a 62 page essay on The Logic of Metabolism which really was quite striking; one of those texts you can’t put down (seems such a long time ago now!).

» Phys.org: Was life inevitable? New paper pieces together metabolism’s beginnings

» Open access arXiv preprint of the work: q-bio/1207.5532

All this just shows the authors know their field however, and from points made by others they build their investigation: are modern biochemical reaction sequences result of evolutionary selection, or was the initial metabolic reaction network a ready-made entity when life popped up?

As much as the tone is towards the latter, the authors concede both possibilities have their individual merit. Firm experimental evidence for either is sorely lacking.

Fundamental experiments

In water, the group observed 17 cases of glycolytic/pentose phosphate pathway (PPP) intermediates being converted into other metabolites.

In the pentose phosphate pathway, we observed isomerization of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, as well as the formation of glyceraldehyde 3-phosphate from these intermediates (Fig. A, left panel). In cellular metabolism, analogous reaction sequences do exist and are catalysed by the pentose phosphate pathway enzymes ribulose 5-phosphate epimerase, ribose 5-phosphate isomerase and transketolase (Fig. D, left panel). Glycolytic intermediates converted into pyruvate and glucose, the stable products of glycolysis and gluconeogenesis, as well as the intermediate metabolite glucose 6-phosphate (Fig. A, right panel).

Overall, pyruvate formation dominated. Its non-enzymatic formation was detected from PPP metabolites, fructose 6-phosphate, fructose 1,6-bisphosphate and all intermediates of lower glycolysis (Fig. D, right panel). It is therefore apparent that heat exposure is sufficient to convert intermediate metabolites of glycolysis and the pentose phosphate pathway into pyruvate and glucose that constitute thermodynamically stable products also in the modern, enzyme-catalysed metabolism, and to induce isomerization between pentose phosphate metabolites.

To clarify in case it may not be obvious having removed the figure caption, these diagrams show metabolic pathways, or ‘network topologies’ in plain old (high-purity ULC-MS grade) water (A), ‘plausible concentrations’ [for the Archean ocean] of Na, Cl, K, BO3, F, PO4, Mg, Ca, Si, Mo, Co, Ni and Fe (B), a ferrous Fe(II)-supplemented version of this Archean mimetic, i.e. maintaining anoxic conditions (C) and lastly the ‘modern’ enzyme-bolstered glycolysis (canonical Embden-Meyerhof) and pentose phosphate pathways.

After looking at water, the group tested whether conditions replicating the pre-oxygenation oceanic environment would influence these reactions, and as shown above they did, significantly. Amazingly (perhaps, though it does sound labour-intensive) they quantified the metabolites in the resultant 1,200 samples by manually supervised LC-MS/MS peak identification and integration of 18,000 resultant chromatographic peaks. Oceanic salts did not show any significant influence on the sugar phosphate interconversions, which is a notable result in itself.

However, a reactive solution containing the metal ions Fe, Co, Ni, Mo as well as phosphate at Archean ocean plausible concentrations catalysed additional reactions. In the metal-rich solution, out of the 182 reactions monitored, we observed in 28 cases the conversion of a glycolytic or pentose phosphate metabolite into another intermediate of the two pathways (Fig. B). The reactions catalysed by the Archean ocean mimetic yielded the formation of ribose 5-phosphate, the constituent of the RNA backbone and erythrose 4-phosphate, the precursor of aromatic amino acids, starting from several pentose phosphate pathway intermediates.

As a result, these non-enzymatic reactions interconnected all pentose phosphate pathway intermediates (glucose 6-phosphate, erythrose 4-phosphate, ribose 5-phosphate, ribulose 5-phosphate, xylulose 5-phosphate and sedoheptulose 7-phosphate). The interconversion network corresponds to the reaction sequence of the modern-cell non-oxidative pentose pathway (Fig. B and D). In difference, interconversion reactions that would correspond to reactions of the oxidative pentose phosphate pathway, converting glucose 6-phosphate to 6-phosphogluconate, were not observed.

This ocean-in-a-test-tube allowed some neat little tests to be carried out with 6-phosphogluconate (6PG), stable at 70°C in water, but reacting to form PPP intermediates (⇒ pyruvate). The sheer scale of these experiments is impressive ‒ they monitored 182 possible interconversion reactions in the iron-rich mimetic. How many trained lab monkeys does it take to watch 182 reactions you might ask — actually the team used LC-SRM, the second half of which stands for Selected Reaction Monitoringanother form of mass spectrometry. It saw some use in these experiments since it’s really sensitive (it can measure PTMs below the range of regular mass spec.) and its ‘data-independent nature’.

So as to give more than just a ‘network topology’, they were also able to show that ferrous iron increased specificity, leading to a greater mean carbon recovery in products (62% vs. 50%), i.e. ¼ of the absolute concentrations of non-enzymatically interconverted metabolites formed an intermediate of glycolysis/PPP (calculated with 62% being around ~125% of 50% in case you were wondering!) — i.e. there was quite a favourable yield in this ‘one-pot’ organic synthesis.

The determined reaction rates ranged over four orders of magnitude. In the ferrous iron-rich Archean ocean mimetic, the fastest observed reaction occurred at a rate 19.6 lM/h and was the conversion 6-phosphogluconate to the RNA backbone precursor ribose 5-phosphate. In contrast, the slowest reaction, the dephosphorylation of glucose 6-phosphate to glucose, occurred at a rate of 0.09 lM/h. Several of the interconversion rates were faster in the Archean ocean mimetics compared to the water (12 out of the 17 reactions detected in water); comparing the mimetics, 12 reactions were accelerated in the presence of Fe(II) over Fe(III). 6-phosphogluconate for instance was not converted to ribose 5-phosphate in water. In the ferric iron-rich mimetic, this reaction was observed at a rate of 5.5 lM/h, while in the ferrous iron-rich anoxic experiment at a rate of 19.6 lM/h. In some cases, acceleration was also observed for the three-carbon phosphate interconversion reactions that are analogous to reactions of lower glycolysis; that is, the conversion of phosphoenolpyruvate to pyruvate occurred at 13.5 lM/h in the Fe(II)-rich Archean ocean mimetic, while a rate of 7.3 lM/h was observed in water. However, several of the of three-carbon metabolite formation reactions, including the formation of pyruvate from fructose 1,6-bisphosphate, or glyceraldehyde 3-phosphate from ribose 5-phosphate, were slowed down, most likely due to the stabilization of precursors.

In order to get a global picture of the reactivity in the system, we therefore combined all intermediate metabolites at a concentration of 7.5 lM and determined the temperature-dependent rate of pyruvate formation. At temperatures of 40°C and below, no pyruvate was formed, confirming the absence of enzymatic contamination in the combined reaction mimetic. Above 50°C, pyruvate formation was detected and increased in a temperature-dependent manner. The fastest pyruvate formation rate was detected at 90°C, where typical metabolic enzymes are not functional. Comparing the three conditions, pyruvate formation rate was lowest in water, increased in the presence of the ocean components and achieved its highest rate in the ferrous iron-rich Archean ocean simulation. Compared to water at a temperature of 70°C, the pyruvate formation was 49% faster in the presence of the Archean ocean mimetic and ferric iron, and 200% faster in the analogous, ferrous iron containing ocean simulation. Hence, the ferrous condition plausible for the Archean ocean does not only catalyse interconversion among pentose phosphate sugars, they also favoured specificity and increased reactivity in a non-enzymatic, glycolysis-like, chemical reaction system.

The final step in proceedings set up a low concentration of the aforementioned mineral ions, with non-biological optimum temperatures ruling out any enzymes having snuck through, and what’s more confirming the ferrous iron increases reactivity of glycosysis-like chemical reactions.

What’s so fascinating about metabolism-first theories is that they still need evolutionary explanation ‒ that is, how did life come to harness these processes, and what did it bring to the table in doing so? Here, the authors note that the evolutionary origins of the Embden-Meyerhof network structure are ‘still largely unknown’.

Geological records reveal details about the chemical environment under which life spread for the first time. This event has been dated between the earliest Archean eon that followed the late heavy bombardment, likely between 4.1 and 3.5 billion years ago, when the first unequivocal traces of life have been dated. Geochemical and geological evidence, particularly the large iron isotope fractionations and the lack of sulphur isotope fractionation during pyrite burial, suggests that the oceans of this period were rich in ferrous iron and poor in sulfate. The absolute concentration of iron in these ancient oceans is equivocal with estimates ranging from 20 lM to 5 mM. Knowledge about the concentrations of other bio-essential metals in the Archean oceans during the evolution of life comes from reconstructing the biogeochemical cycles of these elements and taking into account the environmental properties of a world devoid of oxygen. This has led to the suggestion that copper concentrations in the Archean oceans were negligible and that cobalt and manganese were more abundant than today (but still many orders of magnitude lower than the paleo iron concentrations). The same argument has been applied to zinc; however, its concentration in Proterozoic black shales and Archean iron oxides suggests that its concentration may have remained closer to modern levels over much of the geological time. Nickel and molybdenum concentrations were likely lower than today, whereas concentrations of cobalt and manganese are assumed to be similar to modern levels. It has further been suggested that phosphorus is more easily released from sediment in anoxic (or dysoxic) bottom waters, hinting that the Archean oceans may have been rich in phosphate.

The only flaw in this paper is that in parts it feels a little vague ‒ I don’t think I’ll be the only person reading this wondering exactly what the significance of comparing network topology is, but it could well be my own miseducation showing through. Similarly, the how of the origin of life is sparse on detail, but in fairness this is asking a bit much of one paper.

One of the difficulties in describing the origin of metabolism is the fact that the metabolic network is largely composed of intermediates that are not characterized by long-time stability, at least when considering geological environments and timescales. As shown here and previously, this in particular applies to sugar phosphate molecules. In addition, large sugar phosphates are not frequently generated in experiments that address scenarios of primordial carbon fixation. This difficulty cannot, however, mask the fact that sugar phosphates are constituents of many molecules, such as RNA, DNA, ATP and lipids, which are inevitably connected with the emergence of life. It is the fundamental role of sugar phosphates, and the virtual universality of their few metabolic interconversion sequences, that places their origin to the very early evolutionary stages.

Long-term stability seems thus not to be a predictor of whether a molecule adopts a cellular metabolic function. A possible explanation is that molecules stable in a certain environment do not react with their surrounding molecules; inert molecules were thus unlikely to form reaction systems based on environmental catalysts. This seeming paradox of the universality of sugar phosphates and their low stability in the prebiotic world might be solved by accepting different reaction sequences for carbon fixation and the first forms of metabolism: Carbon fixation could have occurred through non-metabolism-like events, including the so-called formose reaction, which is, in a series of condensation steps, able to convert several formaldehyde molecules into complex carbohydrates structures, or through alternative/parallel scenarios that include mineral- or photochemically catalysed reactions, as well as microcompartmentalization, that allowed the accumulation of first biomolecules in protocells. It is only when the first biomolecules had achieved life-compatible concentrations that biologically relevant interconversion sequences, or early forms of metabolic pathways, could become active.

This seems to place their previous results in a bit of a grey area, given that they were produced with the intention of simulating environmental conditions, but in discussing their ideas on the matter the authors are pointing elsewhere for the first sparks of metabolism. The team are the first to point out what they missed — mineral surfaces can act as catalysts; reaction conditions were not optimised to maximise the number of reactions (rather, the reconstruction ran the other way around).

It is thus equally well possible that this part of the pathway came into being with the emergence of enzymes. Therefore, these results could support a hybrid hypothesis to describe the origin of metabolism: a core set of reactions would have be constrained by the environment of the early world organisms, and this network was then extended in terms of both reactivity and efficiency through the evolutionary selection of enzymes until the modern network was in place.

The array of possibilities is staggering. Lastly, it wouldn’t be an origin of life story without some weird Creature From The Deep, and before finishing the group slip in mention of thermophilic microorganisms that have a functional ‘non-phosphorylating’ Entner-Doudoroff pathway, not yielding any ATP from glucose, just metabolic intermediates. This gives some hope to the possibility of non-ATP-generating glycolysis as described having likewise been a part of the grand scheme of things back in some long-bygone era.

The results presented here demonstrate that sugar phosphate interconversions reactions are prebiotically plausible; thus, the origin of the ribose 5-phosphate through a sugar phosphate interconversion route should be considered. In this context, we have noticed that ribose 5-phosphate was around five times more stable than the other pentose phosphate intermediates (xylulose 5-phosphate and ribulose 5-phosphate) and the formation of ribose 5-phosphate from 6-phosphogluconate was the fastest of all reactions in the presence of Fe(II) (Fig 5A). These properties could have contributed to the central importance of ribose 5-phosphate as backbone of the genetic material.

Looking ahead, the authors propose a deeper inspection of the thermodynamics here, which I look forward to seeing and will fit in nicely with others in the field working towards the same goal.

Studies about the thermodynamics in metabolic systems have, however, shown that reaction equilibrium is shifted under conditions that separate a product from the catalyst, when reactions are coupled and when one product is stabilized, that is, by the inclusion in protocell-like vesicles (Amend et al, 2013). In future studies, it should thus be investigated whether the reactions reported here can be combined with prebiotically plausible mechanisms to stabilize the higher energetic phosphates and thus whether the reactions can be exploited in carbon fixation mechanisms as well.

Update: Larry has now written about this paper himself, and is discussing how he feels it failed to raise contradictions with alternative theories in the comments with co-author Markus Ralser. Interestingly he classifies this as the ‘metabolic soup’ line of thinking, since gluconeogenesis would have sprung up before glycolysis and pentose phosphate pathways.

In particular he seems to feel the authors failed to cite other research (I mean even I knew Nick Lane is one of the eminent scholars in this area yet his work is only cited once, in passing). Food for thought, and a reminder to consider what might be going unsaid. I’ll have to check back after Ralser has replied, could be an interesting discussion.

⇅ Keller MA, Turchyn AV and Ralser M (2014) Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in the plausible Archean ocean. Molecular Systems Biology 10:725

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