Hydrogel-based transparent soils for root phenotyping in vivo.

Root phenotypes are increasingly explored as predictors of crop performance but are still challenging to characterize. Media that mimic field conditions (e.g., soil, sand) are opaque to most forms of radiation, while transparent media do not provide field-relevant growing conditions and phenotypes. We describe here a "transparent soil" formed by the spherification of hydrogels of biopolymers. It is specifically designed to support root growth in the presence of air, water, and nutrients, and allows the time-resolved phenotyping of roots in vivo by both photography and microscopy. The roots developed by soybean plants in this medium are significantly more similar to those developed in real soil than those developed in hydroponic conditions and do not show signs of hypoxia. Lastly, we show that the granular nature and tunable properties of these hydrogel beads can be leveraged to investigate the response of roots to gradients in water availability and soil stiffness.

Porcine signal regulatory protein alpha binds to human CD47 to inhibit phagocytosis: Implications for human hematopoietic stem cell transplantation into severe combined immunodeficient pigs.

BACKGROUND: Severe combined immunodeficient (SCID) pigs are an emerging animal model being developed for biomedical and regenerative medicine research. SCID pigs can successfully engraft human-induced pluripotent stem cells and cancer cell lines. The development of a humanized SCID pig through xenotransplantation of human hematopoietic stem cells (HSCs) would be a further demonstration of the value of such a large animal SCID model. Xenotransplantation success with HSCs into non-obese diabetic (NOD)-derived SCID mice is dependent on the ability of NOD mouse signal regulatory protein alpha (SIRPA) to bind human CD47, inducing higher phagocytic tolerance than other mouse strains. Therefore, we investigated whether porcine SIRPA binds human CD47 in the context of developing a humanized SCID pig. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from SCID and non-SCID pigs. Flow cytometry was used to assess whether porcine monocytes could bind to human CD47. Porcine monocytes were isolated from PBMCs and were subjected to phagocytosis assays with pig, human, and mouse red blood cell (RBC) targets. Blocking phagocytosis assays were performed by incubating human RBCs with anti-human CD47 blocking antibody B6H12, non-blocking antibody 2D3, and nonspecific IgG1 antibody and exposing to human or porcine monocytes. RESULTS: We found that porcine SIRPA binds to human CD47 in vitro by flow cytometric assays. Additionally, phagocytosis assays were performed, and we found that porcine monocytes phagocytose human and porcine RBCs at significantly lower levels than mouse RBCs. When human RBCs were preincubated with CD47 antibodies B6H12 or 2D3, phagocytosis was induced only after B6H12 incubation, indicating the lower phagocytic activity of porcine monocytes with human cells requires interaction between porcine SIRPA and human CD47. CONCLUSIONS: We have shown the first evidence that porcine monocytes can bind to human CD47 and are phagocytically tolerant to human cells, suggesting that porcine SCID models have the potential to support engraftment of human HSCs.

Active Site Breathing of Human Alkbh5 Revealed by Solution NMR and Accelerated Molecular Dynamics.

AlkB homolog 5 (Alkbh5) is one of nine members of the AlkB family, which are nonheme Fe2+/α-ketoglutarate-dependent dioxygenases that catalyze the oxidative demethylation of modified nucleotides and amino acids. Alkbh5 is highly selective for the N6-methyladenosine modification, an epigenetic mark that has spawned significant biological and pharmacological interest because of its involvement in important physiological processes, such as carcinogenesis and stem cell differentiation. Herein, we investigate the structure and dynamics of human Alkbh5 in solution. By using 15N and 13Cmethyl relaxation dispersion and 15N-R1 and R1ρ NMR experiments, we show that the active site of apo Alkbh5 experiences conformational dynamics on multiple timescales. Consistent with this observation, backbone amide residual dipolar couplings measured for Alkbh5 in phage pf1 are inconsistent with the static crystal structure of the enzyme. We developed a simple approach that combines residual dipolar coupling data and accelerated molecular dynamics simulations to calculate a conformational ensemble of Alkbh5 that is fully consistent with the experimental NMR data. Our structural model reveals that Alkbh5 is more disordered in solution than what is observed in the crystal state and undergoes breathing motions that expand the active site and allow access to α-ketoglutarate. Disordered-to-ordered conformational changes induced by sequential substrate/cofactor binding events have been often invoked to interpret biochemical data on the activity and specificity of AlkB proteins. The structural ensemble reported in this work provides the first atomic-resolution model of an AlkB protein in its disordered conformational state to our knowledge.

Hybrid Approaches to Structural Characterization of Conformational Ensembles of Complex Macromolecular Systems Combining NMR Residual Dipolar Couplings and Solution X-ray Scattering.

Solving structures or structural ensembles of large macromolecular systems in solution poses a challenging problem. While NMR provides structural information at atomic resolution, increased spectral complexity, chemical shift overlap, and short transverse relaxation times (associated with slow tumbling) render application of the usual techniques that have been so successful for medium sized systems (<50 kDa) difficult. Solution X-ray scattering, on the other hand, is not limited by molecular weight but only provides low resolution structural information related to the overall shape and size of the system under investigation. Here we review how combining atomic resolution structures of smaller domains with sparse experimental data afforded by NMR residual dipolar couplings (which yield both orientational and shape information) and solution X-ray scattering data in rigid-body simulated annealing calculations provides a powerful approach for investigating the structural aspects of conformational dynamics in large multidomain proteins. The application of this hybrid methodology is illustrated for the 128 kDa dimer of bacterial Enzyme I which exists in a variety of open and closed states that are sampled at various points in the catalytic cycles, and for the capsid protein of the human immunodeficiency virus.

Mechanistic Insight into Nanoparticle Surface Adsorption by Solution NMR Spectroscopy in an Aqueous Gel.

Engineering nanoparticle (NP) functions at the molecular level requires a detailed understanding of the dynamic processes occurring at the NP surface. Herein we show that a combination of dark-state exchange saturation transfer (DEST) and relaxation dispersion (RD) NMR experiments on gel-stabilized NP samples enables the accurate determination of the kinetics and thermodynamics of adsorption. We used the former approach to describe the interaction of cholic acid (CA) and phenol (PhOH) with ceria NPs with a diameter of approximately 200 nm. Whereas CA formed weak interactions with the NPs, PhOH was tightly bound to the NP surface. Interestingly, we found that the adsorption of PhOH proceeds via an intermediate, weakly bound state in which the small molecule has residual degrees of rotational diffusion. We believe the use of aqueous gels for stabilizing NP samples will increase the applicability of solution NMR methods to the characterization of nanomaterials.