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

A chromatin modifying enzyme, SDG8, is involved in morphological, gene expression, and epigenetic responses to mechanical stimulation

Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzyme, SDG8/ASHH2, which can regulate the expression of many touch responsive genes identified in Arabidopsis. SDG8 is required for the permissive expression of touch induced genes; and the loss of function of sdg8 perturbs the maximum levels of induction on selected touch gene targets. SDG8 is required to maintain permissive H3K4 trimethylation marks surrounding the Arabidopsis touch-inducible gene TOUCH 3 (TCH3), which encodes a calmodulin-like protein (CML12). The gene neighboring was also slightly down regulated, revealing a new target for SDG8 mediated chromatin modification. Finally, sdg8 mutants show perturbed morphological response to wind-agitated mechanical stimuli, implicating an epigenetic memory-forming process in the acclimation response of thigmomorphogenesis

A Framework for Evaluating Approximation Methods for Gaussian Process Regression

Gaussian process (GP) predictors are an important component of many Bayesian approaches to machine learning. However, even a straightforward implementation of Gaussian process regression (GPR) requires O(n^2) space and O(n^3) time for a data set of n examples. Several approximation methods have been proposed, but there is a lack of understanding of the relative merits of the different approximations, and in what situations they are most useful. We recommend assessing the quality of the predictions obtained as a function of the compute time taken, and comparing to standard baselines (e.g., Subset of Data and FITC). We empirically investigate four different approximation algorithms on four different prediction problems, and make our code available to encourage future comparisons

Lack of association between the rs2294008 polymorphism in the prostate stem cell antigen gene and colorectal neoplasia : a case-control and immunohistochemical study

PMID: 22824379 [PubMed - indexed for MEDLINE] PMCID: PMC3500224 Free PMC ArticlePeer reviewedPublisher PD

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

Predicting First Traversal Times for Virions and Nanoparticles in Mucus with Slowed Diffusion

AbstractParticle-tracking experiments focusing on virions or nanoparticles in mucus have measured mean-square displacements and reported diffusion coefficients that are orders of magnitude smaller than the diffusion coefficients of such particles in water. Accurate description of this subdiffusion is important to properly estimate the likelihood of virions traversing the mucus boundary layer and infecting cells in the epithelium. However, there are several candidate models for diffusion that can fit experimental measurements of mean-square displacements. We show that these models yield very different estimates for the time taken for subdiffusive virions to traverse through a mucus layer. We explain why fits of subdiffusive mean-square displacements to standard diffusion models may be misleading. Relevant to human immunodeficiency virus infection, using computational methods for fractional subdiffusion, we show that subdiffusion in normal acidic mucus provides a more effective barrier against infection than previously thought. By contrast, the neutralization of the mucus by alkaline semen, after sexual intercourse, allows virions to cross the mucus layer and reach the epithelium in a short timeframe. The computed barrier protection from fractional subdiffusion is some orders of magnitude greater than that derived by fitting standard models of diffusion to subdiffusive data

Model Criticism in Latent Space

Model criticism is usually carried out by assessing if replicated data generated under the fitted model looks similar to the observed data, see e.g. Gelman, Carlin, Stern, and Rubin [2004, p. 165]. This paper presents a method for latent variable models by pulling back the data into the space of latent variables, and carrying out model criticism in that space. Making use of a model's structure enables a more direct assessment of the assumptions made in the prior and likelihood. We demonstrate the method with examples of model criticism in latent space applied to factor analysis, linear dynamical systems and Gaussian processes

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

XBootes: An X-Ray Survey of the NDWFS Bootes Field - Paper I Overview and Initial Results

We obtained a 5 ksec deep Chandra X-ray Observatory ACIS-I map of the 9.3 square degree Bootes field of the NOAO Deep Wide-Field Survey. Here we describe the data acquisition and analysis strategies leading to a catalog of 4642 (3293) point sources with 2 or more (4 or more) counts, corresponding to a limiting flux of roughly 4(8)x10^{-15} erg cm^{-2}s^{-1} in the 0.5-7 keV band. These Chandra XBootes data are unique in that they consitute the widest contiguous X-ray field yet observed to such a faint flux limit. Because of the extraordinarily low background of the ACIS, we expect only 14% (0.7%) of the sources to be spurious. We also detected 43 extended sources in this survey. The distribution of the point sources among the 126 pointings (ACIS-I has a 16 x 16 arcminute field of view) is consistent with Poisson fluctuations about the mean of 36.8 sources per pointing. While a smoothed image of the point source distribution is clumpy, there is no statistically significant evidence of large scale filamentary structure. We do find however, that for theta>1 arcminute, the angular correlation function of these sources is consistent with previous measurements, following a power law in angle with slope -0.7. In a 1.4 deg^{2} sample of the survey, approximately 87% of the sources with 4 or more counts have an optical counterpart to R ~26 mag. As part of a larger program of optical spectroscopy of the NDWFS Bootes area, spectra have been obtained for \~900 of the X-ray sources, most of which are QSOs or AGN.Comment: 18 Pages, 10 figures (AASTex Preprint format