Combinatorial biomaterials discovery strategy to identify new macromolecular cryoprotectants

Cryoprotective agents (CPAs) are typically solvents or small molecules, but there is a need for innovative CPAs to reduce toxicity and increase cell yield, for the banking and transport of cells. Here we use a photochemical high-throughput discovery platform to identify macromolecular cryoprotectants, as rational design approaches are currently limited by the lack of structure–property relationships. Using liquid handling systems, 120 unique polyampholytes were synthesized using photopolymerization with RAFT agents. Cryopreservation screening identified “hit” polymers and nonlinear trends between composition and function, highlighting the requirement for screening, with polymer aggregation being a key factor. The most active polymers reduced the volume of dimethyl sulfoxide (DMSO) required to cryopreserve a nucleated cell line, demonstrating the potential of this approach to identify materials for cell storage and transport

Regioregular alternating polyampholytes have enhanced biomimetic ice recrystallization activity compared to random copolymers and the role of side chain verses main chain hydrophobicity

Antifreeze proteins from polar fish species are potent ice recrystallization inhibitors (IRIs) effectively stopping all ice growth. Additives which have IRI activity have been shown to enhance cellular cryopreservation with potential to improve the distribution of donor cells and tissue. Polyampholytes, polymers with both anionic and cationic side chains, are a rapidly emerging class of polymer cryoprotectants, but their mode of action and the structural features essential for activity are not clear. Here regio-regular polyampholytes are synthesized from maleic anhydride co-polymers to enable stoichiometric installation of the charged groups, ensuring regio-regularity which is not possible using conventional random co-polymerisation. A modular synthetic strategy is employed to enable the backbone and side chain hydrophobicity to be varied, with side chain hydrophobicity found to have a profound effect on the IRI activity. The activity of the regio-regular polymers was found to be superior to those derived from a standard random copolymerisation with statistical incorporation of monomers, demonstrating that sequence composition is crucial to the activity of IRI active polyampholytes

Polyampholytes as emerging macromolecular cryoprotectants

Cellular cryopreservation is a platform technology which underpins cell biology, biochemistry, biomaterials, diagnostics, and the cold chain for emerging cell-based therapies. This technique relies on effective methods for banking and shipping to avoid the need for continuous cell culture. The most common method to achieve cryopreservation is to use large volumes of organic solvent cryoprotective agents which can promote either a vitreous (ice free) phase or dehydrate and protect the cells. These methods are very successful but are not perfect: not all cell types can be cryopreserved and recovered, and the cells do not always retain their phenotype and function post-thaw. This Perspective will introduce polyampholytes as emerging macromolecular cryoprotective agents and demonstrate they have the potential to impact a range of fields from cell-based therapies to basic cell biology and may be able to improve, or replace, current solvent-based cryoprotective agents. Polyampholytes have been shown to be remarkable (mammalian cell) cryopreservation enhancers, but their mechanism of action is unclear, which may include membrane protection, solvent replacement, or a yet unknown protective mechanism, but it seems the modulation of ice growth (recrystallization) may only play a minor role in their function, unlike other macromolecular cryoprotectants. This Perspective will discuss their synthesis and summarize the state-of-the-art, including hypotheses of how they function, to introduce this exciting area of biomacromolecular science

Public Domain Treaty Compliance Verification in the Digital Age

We explore in this article some of the emerging opportunities, and associated challenges, that the digital age offers for public-domain verification of compliance with international treaties. The increase in data volume, in ever-improving connectivity, and the relentless evolution towards ubiquitous sensors all provide a rapidly changing landscape for technical compliance verification of international treaties. From satellites to cell phones, advances in technology afford new opportunities for verifying compliance with international agreements, on topics ranging from arms control to environmental and public health issues. We will identify some of the engineering challenges that must be overcome in order to realize these new verification opportunities.Physic

Uniformity of V minus Near Infrared Color Evolution of Type Ia Supernovae, and Implications for Host Galaxy Extinction Determination

From an analysis of SNe 1972E, 1980N, 1981B, 1981D, 1983R, 1998bu, 1999cl, and 1999cp we find that the intrinsic V-K colors of Type Ia SNe with multi-color light curve shape (MLCS) parameter -0.4 < Delta < +0.2 suggest a uniform color curve. V-K colors become bluer linearly with time from roughly one week before B-band maximum until one week after maximum, after which they redden linearly until four weeks after maximum. V-H colors exhibit very similar color evolution. V-J colors exhibit slightly more complex evolution, with greater scatter. The existence of V minus near infrared color relations allows the construction of near infrared light curve templates that are an improvement on those of Elias et al. (1985). We provide optical BVRI and infrared JHK photometry of the Type Ia supernovae 1999aa, 1999cl, and 1999cp. SN 1999aa is an overluminous "slow decliner" (with Delta = -0.47 mag). SN 1999cp is a moderately bright SN unreddened in its host. SN 1999cl is extremely reddened in its host. The V minus near infrared colors of SN 1999cl yield A_V = 2.01 +/- 0.11 mag. This leads to a distance for its host galaxy (M 88) in agreement with other distance measurements for members of the Virgo cluster.Comment: 57 pages, 13 postscript figures, to appear in the August 20, 2000, issue of the Astrophysical Journal. Contains updated references and a number of minor corrections dealt with when page proofs were correcte

Enhancement of macromolecular ice recrystallization inhibition activity by exploiting depletion forces

Antifreeze (glyco) proteins (AF(G)Ps) are potent inhibitors of ice recrystallization and may have biotechnological applications. The most potent AF(G)Ps function at concentrations a thousand times lower than synthetic mimics such as poly(vinyl alcohol), PVA. Here, we demonstrate that PVA’s ice recrystallization activity can be rescued at concentrations where it does not normally function, by the addition of noninteracting polymeric depletants, due to PVA forming colloids in the concentrated saline environment present between ice crystals. These depletants shift the equilibrium toward ice binding and, hence, enable PVA to inhibit ice growth at lower concentrations. Using theory and experiments, we show this effect requires polymeric depletants, not small molecules, to enhance activity. These results increase our understanding of how to design new ice growth inhibitors, but also offer opportunities to enhance activity by exploiting depletion forces, without re-engineering ice-binding materials. It also shows that when screening for IRI activity that polymer contaminants in buffers may give rise to false positive results

Solar system constraints on the Dvali-Gabadadze-Porrati braneworld theory of gravity

A number of proposals have been put forward to account for the observed accelerating expansion of the Universe through modifications of gravity. One specific scenario, Dvali-Gabadadze-Porrati (DGP) gravity, gives rise to a potentially observable anomaly in the solar system: all planets would exhibit a common anomalous precession, dw/dt, in excess of the prediction of General Relativity. We have used the Planetary Ephemeris Program (PEP) along with planetary radar and radio tracking data to set a constraint of |dw/dt| < 0.02 arcseconds per century on the presence of any such common precession. This sensitivity falls short of that needed to detect the estimated universal precession of |dw/dt| = 5e-4 arcseconds per century expected in the DGP scenario. We discuss the fact that ranging data between objects that orbit in a common plane cannot constrain the DGP scenario. It is only through the relative inclinations of the planetary orbital planes that solar system ranging data have sensitivity to the DGP-like effect of universal precession. In addition, we illustrate the importance of performing a numerical evaluation of the sensitivity of the data set and model to any perturbative precession.Comment: 9 pages, 2 figures, accepted for publication in Phys. Rev.

Synthetically scalable poly(ampholyte) which dramatically enhances cellular cryopreservation

The storage and transport of frozen cells underpin the emerging/existing cell-based therapies and are used in every biomedical research lab globally. The current gold-standard cryoprotectant dimethyl sulfoxide (DMSO) does not give quantitative cell recovery in suspension or in two-dimensional (2D) or three-dimensional (3D) cell models, and the solvent and cell debris must be removed prior to application/transfusion. There is a real need to improve this 50-year-old method to underpin emerging regenerative and cell-based therapies. Here, we introduce a potent and synthetically scalable polymeric cryopreservation enhancer which is easily obtained in a single step from a low cost and biocompatible precursor, poly(methyl vinyl ether-alt-maleic anhydride). This poly(ampholyte) enables post-thaw recoveries of up to 88% for a 2D cell monolayer model compared to just 24% using conventional DMSO cryopreservation. The poly(ampholyte) also enables reduction of [DMSO] from 10 wt % to just 2.5 wt % in suspension cryopreservation, which can reduce the negative side effects and speed up post-thaw processing. After thawing, the cells have reduced membrane damage and faster growth rates compared to those without the polymer. The polymer appears to function by a unique extracellular mechanism by stabilization of the cell membrane, rather than by modulation of ice formation and growth. This new macromolecular cryoprotectant will find applications across basic and translational biomedical science and may improve the cold chain for cell-based therapies

Equivalence Principle Implications of Modified Gravity Models

Theories that attempt to explain the observed cosmic acceleration by modifying general relativity all introduce a new scalar degree of freedom that is active on large scales, but is screened on small scales to match experiments. We show that if such screening occurrs via the chameleon mechanism such as in f(R), it is possible to have order one violation of the equivalence principle, despite the absence of explicit violation in the microscopic action. Namely, extended objects such as galaxies or constituents thereof do not all fall at the same rate. The chameleon mechanism can screen the scalar charge for large objects but not for small ones (large/small is defined by the gravitational potential and controlled by the scalar coupling). This leads to order one fluctuations in the inertial to gravitational mass ratio. In Jordan frame, it is no longer true that all objects move on geodesics. In contrast, if the scalar screening occurrs via strong coupling, such as in the DGP braneworld model, equivalence principle violation occurrs at a much reduced level. We propose several observational tests of the chameleon mechanism: 1. small galaxies should fall faster than large galaxies, even when dynamical friction is negligible; 2. voids defined by small galaxies would be larger compared to standard expectations; 3. stars and diffuse gas in small galaxies should have different velocities, even on the same orbits; 4. lensing and dynamical mass estimates should agree for large galaxies but disagree for small ones. We discuss possible pitfalls in some of these tests. The cleanest is the third one where mass estimate from HI rotational velocity could exceed that from stars by 30 % or more. To avoid blanket screening of all objects, the most promising place to look is in voids.Comment: 27 pages, 4 figures, minor revisions, references added. Accepted for publication in Phys. Rev.

The atomistic details of the ice recrystallisation inhibition activity of PVA

Understanding the ice recrystallisation inhibition (IRI) activity of antifreeze biomimetics is crucial to the development of the next generation of cryoprotectants. In this work, we bring together molecular dynamics simulations and quantitative experimental measurements to unravel the microscopic origins of the IRI activity of poly(vinyl)alcohol (PVA)—the most potent of biomimetic IRI agents. Contrary to the emerging consensus, we find that PVA does not require a “lattice matching” to ice in order to display IRI activity: instead, it is the effective volume of PVA and its contact area with the ice surface which dictates its IRI strength. We also find that entropic contributions may play a role in the ice-PVA interaction and we demonstrate that small block co-polymers (up to now thought to be IRI-inactive) might display significant IRI potential. This work clarifies the atomistic details of the IRI activity of PVA and provides novel guidelines for the rational design of cryoprotectants