Biomanufacturing in low Earth orbit for regenerative medicine.

Research in low Earth orbit (LEO) has become more accessible. The 2020 Biomanufacturing in Space Symposium reviewed space-based regenerative medicine research and discussed leveraging LEO to advance biomanufacturing for regenerative medicine applications. The symposium identified areas where financial investments could stimulate advancements overcoming technical barriers. Opportunities in disease modeling, stem-cell-derived products, and biofabrication were highlighted. The symposium will initiate a roadmap to a sustainable market for regenerative medicine biomanufacturing in space. This perspective summarizes the 2020 Biomanufacturing in Space Symposium, highlights key biomanufacturing opportunities in LEO, and lays the framework for a roadmap to regenerative medicine biomanufacturing in space.

Activation of retinal ganglion cells using a biomimetic artificial retina.

Objective.Biomimetic protein-based artificial retinas offer a new paradigm for restoring vision for patients blinded by retinal degeneration. Artificial retinas, comprised of an ion-permeable membrane and alternating layers of bacteriorhodopsin (BR) and a polycation binder, are assembled using layer-by-layer electrostatic adsorption. Upon light absorption, the oriented BR layers generate a unidirectional proton gradient. The main objective of this investigation is to demonstrate the ability of the ion-mediated subretinal artificial retina to activate retinal ganglion cells (RGCs) of degenerated retinal tissue.Approach. Ex vivoextracellular recording experiments with P23H line 1 rats are used to measure the response of RGCs following selective stimulation of our artificial retina using a pulsed light source. Single-unit recording is used to evaluate the efficiency and latency of activation, while a multielectrode array (MEA) is used to assess the spatial sensitivity of the artificial retina films.Main results.The activation efficiency of the artificial retina increases with increased incident light intensity and demonstrates an activation latency of ∼150 ms. The results suggest that the implant is most efficient with 200 BR layers and can stimulate the retina using light intensities comparable to indoor ambient light. Results from using an MEA show that activation is limited to the targeted receptive field.Significance.The results of this study establish potential effectiveness of using an ion-mediated artificial retina to restore vision for those with degenerative retinal diseases, including retinitis pigmentosa.

The Forbidden 11Bu- Excited Singlet State in Peridinin and Peridinin Analogues.

Theoretical studies have predicted the presence of a forbidden 11Bu- state in proximity to the strongly allowed 11Bu+ excited state in polyenes and carotenoids. The 11Bu- state is invariably predicted to have a very low oscillator strength, which precludes direct optical spectroscopic assignment. We report here a direct UV-vis optical spectroscopic feature assigned to the 11Bu- state of S-2-peridinin, a synthetic analogue of the naturally occurring carotenoid, peridinin. The shift of the ground state dipole of S-2-peridinin compared to natural peridinin enhances the oscillator strength of absorption from the ground state to the 11Bu- state by 2 orders of magnitude relative to peridinin. It is postulated that this is due to a quadrupolar electrostatic field generated from the more central location of the lactone ring along the polyene chain in S-2-peridinin. MNDO-PSDCI and EOM-CCSD calculations provide a theoretical basis for this assignment and explain the unique properties of the 11Bu- state and why the transition from the ground state to this state has such a low oscillator strength in most other polyenes and carotenoids.

Photochromic bacteriorhodopsin mutant with high holographic efficiency and enhanced stability via a putative self-repair mechanism.

The Q photoproduct of bacteriorhodopsin (BR) is the basis of several biophotonic technologies that employ BR as the photoactive element. Several blue BR (bBR) mutants, generated by using directed evolution, were investigated with respect to the photochemical formation of the Q state. We report here a new bBR mutant, D85E/D96Q, which is capable of efficiently converting the entire sample to and from the Q photoproduct. At pH 8.5, where Q formation is optimal, the Q photoproduct requires 65 kJ mol(-1) of amber light irradiation (590 nm) for formation and 5 kJ mol(-1) of blue light (450 nm) for reversion, respectively. The melting temperature of the resting state and Q photoproduct, measured via differential scanning calorimetry, is observed at 100 °C and 89 °C at pH 8.5 or 91 °C and 82 °C at pH 9.5, respectively. We hypothesize that the protein stability of D85E/D96Q compared to other blue mutants is associated with a rapid equilibrium between the blue form E85(H) and the purple form E85(-) of the protein, the latter providing enhanced structural stability. Additionally, the protein is shown to be stable and functional when suspended in an acrylamide matrix at alkaline pH. Real-time photoconversion to and from the Q state is also demonstrated with the immobilized protein. Finally, the holographic efficiency of an ideal thin film using the Q state of D85E/D96Q is calculated to be 16.7%, which is significantly better than that provided by native BR (6-8%) and presents the highest efficiency of any BR mutant to date.

Low-Temperature Trapping of Photointermediates of the Rhodopsin E181Q Mutant.

Three active-site components in rhodopsin play a key role in the stability and function of the protein: 1) the counter-ion residues which stabilize the protonated Schiff base, 2) water molecules, and 3) the hydrogen-bonding network. The ionizable residue Glu-181, which is involved in an extended hydrogen-bonding network with Ser-186, Tyr-268, Tyr-192, and key water molecules within the active site of rhodopsin, has been shown to be involved in a complex counter-ion switch mechanism with Glu-113 during the photobleaching sequence of the protein. Herein, we examine the photobleaching sequence of the E181Q rhodopsin mutant by using cryogenic UV-visible spectroscopy to further elucidate the role of Glu-181 during photoactivation of the protein. We find that lower temperatures are required to trap the early photostationary states of the E181Q mutant compared to native rhodopsin. Additionally, a Blue Shifted Intermediate (BSI, λmax = 498 nm, 100 K) is observed after the formation of E181Q Bathorhodopsin (Batho, λmax = 556 nm, 10 K) but prior to formation of E181Q Lumirhodopsin (Lumi, λmax = 506 nm, 220 K). A potential energy diagram of the observed photointermediates suggests the E181Q Batho intermediate has an enthalpy value 7.99 KJ/mol higher than E181Q BSI, whereas in rhodopsin, the BSI is 10.02 KJ/mol higher in enthalpy than Batho. Thus, the Batho to BSI transition is enthalpically driven in E181Q and entropically driven in native rhodopsin. We conclude that the substitution of Glu-181 with Gln-181 results in a significant perturbation of the hydrogen-bonding network within the active site of rhodopsin. In addition, the removal of a key electrostatic interaction between the chromophore and the protein destabilizes the protein in both the dark state and Batho intermediate conformations while having a stabilizing effect on the BSI conformation. The observed destabilization upon this substitution further supports that Glu-181 is negatively charged in the early intermediates of the photobleaching sequence of rhodopsin.

Directed evolution of bacteriorhodopsin for applications in bioelectronics.

In nature, biological systems gradually evolve through complex, algorithmic processes involving mutation and differential selection. Evolution has optimized biological macromolecules for a variety of functions to provide a comparative advantage. However, nature does not optimize molecules for use in human-made devices, as it would gain no survival advantage in such cooperation. Recent advancements in genetic engineering, most notably directed evolution, have allowed for the stepwise manipulation of the properties of living organisms, promoting the expansion of protein-based devices in nanotechnology. In this review, we highlight the use of directed evolution to optimize photoactive proteins, with an emphasis on bacteriorhodopsin (BR), for device applications. BR, a highly stable light-activated proton pump, has shown great promise in three-dimensional optical memories, real-time holographic processors and artificial retinas.

The nature of the intramolecular charge transfer state in peridinin.

Experimental and theoretical evidence is presented that supports the theory that the intramolecular charge transfer (ICT) state of peridinin is an evolved state formed via excited-state bond-order reversal and solvent reorganization in polar media. The ICT state evolves in <100 fs and is characterized by a large dipole moment (~35 D). The charge transfer character involves a shift of electron density within the polyene chain, and it does not involve participation of molecular orbitals localized in either of the ß-rings. Charge is moved from the allenic side of the polyene into the furanic ring region and is accompanied by bond-order reversal in the central portion of the polyene chain. The electronic properties of the ICT state are generated via mixing of the "1(1)Bu(+)" ionic state and the lowest-lying "2(1)Ag(-)" covalent state. The resulting ICT state is primarily (1)Bu(+)-like in character and exhibits not only a large oscillator strength but an unusually large doubly excited character. In most solvents, two populations exist in equilibrium, one with a lowest-lying ICT ionic state and a second with a lowest-lying "2(1)Ag(-)" covalent state. The two populations are separated by a small barrier associated with solvent relaxation and cavity formation.

Energetics and dynamics of the low-lying electronic states of constrained polyenes: implications for infinite polyenes.

Steady-state and ultrafast transient absorption spectra were obtained for a series of conformationally constrained, isomerically pure polyenes with 5-23 conjugated double bonds (N). These data and fluorescence spectra of the shorter polyenes reveal the N dependence of the energies of six (1)B(u)(+) and two (1)A(g)(-) excited states. The (1)B(u)(+) states converge to a common infinite polyene limit of 15,900 ± 100 cm(-1). The two excited (1)A(g)(-) states, however, exhibit a large (~9000 cm(-1)) energy difference in the infinite polyene limit, in contrast to the common value previously predicted by theory. EOM-CCSD ab initio and MNDO-PSDCI semiempirical MO theories account for the experimental transition energies and intensities. The complex, multistep dynamics of the 1(1)B(u)(+) → 2(1)A(g)(-) → 1(1)A(g)(-) excited state decay pathways as a function of N are compared with kinetic data from several natural and synthetic carotenoids. Distinctive transient absorption signals in the visible region, previously identified with S* states in carotenoids, also are observed for the longer polyenes. Analysis of the lifetimes of the 2(1)A(g)(-) states, using the energy gap law for nonradiative decay, reveals remarkable similarities in the N dependence of the 2(1)A(g)(-) decay kinetics of the carotenoid and polyene systems. These findings are important for understanding the mechanisms by which carotenoids carry out their roles as light-harvesting molecules and photoprotective agents in biological systems.

Effect of Molecular Symmetry on the Spectra and Dynamics of the Intramolecular Charge Transfer (ICT) state of peridinin.

The spectroscopic properties and dynamics of the excited states of two different synthetic analogues of peridinin were investigated as a function of solvent polarity using steady-state absorption, fluorescence, and ultrafast time-resolved optical spectroscopy. The analogues are denoted S-1- and S-2-peridinin and differ from naturally occurring peridinin in the location of the lactone ring and its associated carbonyl group, known to be obligatory for the observation of a solvent dependence of the lifetime of the S(1) state of carotenoids. Relative to peridinin, S-1- and S-2-peridinin have their lactone rings two and four carbons more toward the center of the π-electron system of conjugated carbon-carbon double bonds, respectively. The present experimental results show that as the polarity of the solvent increases, the steady-state spectra of the molecules broaden, and the lowest excited state lifetime of S-1-peridinin changes from ∼155 to ∼17 ps which is similar to the magnitude of the effect reported for peridinin. The solvent-induced change in the lowest excited state lifetime of S-2-peridinin is much smaller and changes only from ∼90 to ∼67 ps as the solvent polarity is increased. These results are interpreted in terms of an intramolecular charge transfer (ICT) state that is formed readily in peridinin and S-1-peridinin, but not in S-2-peridinin. Quantum mechanical computations reveal the critical factors required for the formation of the ICT state and the associated solvent-modulated effects on the spectra and dynamics of these molecules and other carbonyl-containing carotenoids and polyenes. The factors are the magnitude and orientation of the ground- and excited-state dipole moments which must be suitable to generate sufficient mixing of the lowest two excited singlet states.

The intramolecular charge transfer state in carbonyl-containing polyenes and carotenoids.

Numerous femtosecond time-resolved optical spectroscopic experiments have reported that the lifetime of the low-lying S(1) state of carbonyl-containing polyenes and carotenoids decreases with increasing solvent polarity. The effect becomes even more pronounced as the number of double bonds in the conjugated π-electron system decreases. The effect has been attributed to an intramolecular charge transfer (ICT) state coupled to S(1), but it is still not clear what the precise molecular nature of this state is, and how it is able to modulate the spectral and dynamic properties of polyenes and carotenoids. In this work, we examine the nature of the ICT state in three substituted polyenes: crocetindial, which contains two terminal, symmetrically substituted carbonyl groups in conjugation with the π-electron system, 8,8'-diapocarotene-8'-ol-8-al, which has one terminal conjugated carbonyl group and one hydroxyl group, and 8,8'-diapocarotene-8,8'-diol, which has two terminal, symmetrically positioned, hydroxyl groups but no carbonyls. Femtosecond time-resolved optical spectroscopic experiments on these molecules reveal that only the asymmetrically substituted 8,8'-diapocarotene-8'-ol-8-al exhibits any substantial effect of solvent on the excited state spectra and dynamics. The data are interpreted using molecular orbital theory which shows that the ICT state develops via mixing of the low-lying S(1) (2(1)A(g)-like) and S(2) (1(1)B(u)-like) excited singlet states to form a resultant state that preferentially evolves in polar solvent and exhibits a very large (∼25 D) dipole moment. Molecular dynamics calculations demonstrate that the features of the ICT state are present in ∼20 fs.

Rhodium-catalyzed, regiospecific functionalization of polyolefins in the melt.

The rhodium-catalyzed, terminal-selective borylation of alkanes has been used to modify polyolefins. The functionalization of two materials, polyethylethylene (PEE) of molecular weights 1200 and 37 000, was conducted by combining bis-pinacoldiboron and 2.5 mol % [Cp*RhCl2]2 in neat polymer and heating at 150 degrees C. This procedure causes the polymer and boron reagent to melt, the catalyst to dissolve, and the reaction to form material with boryl groups at the terminal position of the polymer side chains. Oxidation of the borylated material generated polymers with hydroxyl groups at the terminal position of the side chains. The functionalization was conducted at various ratios of boron reagent to monomer. The resulting borylated and subsequent hydoxylated materials were characterized by 1H and 13C NMR spectroscopy, as well as MALDI-MS and GPC. Little change in polymer molecular weight and polydispersity was observed, and these data indicate that scission of the main chain does not occur. Measurements of the Tg of the polymers showed in increase in Tg of up to 50 degrees C after the modification. Thus, homogeneous, catalytic, selective alkane functionalization can be used to modify polymer properties.