Investigation of Controlled Growth of Metal-Organic Frameworks on Anisotropic Virus Particles.

Biomimetic mineralization with metal-organic frameworks (MOF), typically zeolitic imidazolate framework-8 (ZIF-8), is an emerging strategy to protect sensitive biological substances against denaturing environmental stressors such as heat and proteolytic agents. Additionally, this same biomimetic mineralization process has the potential of being used to create distinct core-shell architectures using genetically or chemically modified viral nanoparticles. Despite the proliferation of examples for ZIF-8 growth on biological or proteinaceous substrates, systematic studies of these processes are few and far between. Herein, we employed the tobacco mosaic virus (TMV) as a model biological template to investigate the biomimetic mineralization of ZIF-8, which has been proven to be a robust MOF for encasing and protecting inlaid biological substances. Our study shows a systematic dependence upon ZIF-8 crystallization parameters, e.g., ligand to metal molar ratio and metal concentration, which can yield several distinct morphologies of TMV@ZIF-8 composites and phases of ZIF-8. Further investigation using charged synthetic conjugates, time dependent growth analysis, and calorimetric analysis has shown that the TMV-Zn interaction plays a pivotal role in the final morphology of the TMV@ZIF-8, which can take the form of either core-shell bionanoparticles or large crystals of ZIF-8 with entrapped TMV located exclusively on the outer facets. The design rules outlined here, it is hoped, will provide guidance in biomimetic mineralization of MOFs on proteinaceous materials using ZIF-8.

Two-cell embryos are more sensitive than blastocysts to AMPK-dependent suppression of anabolism and stemness by commonly used fertility drugs, a diet supplement, and stress.

PURPOSE: This study tests whether metformin or diet supplement BR-DIM-induced AMP-activated protein kinase (AMPK) mediated effects on development are more pronounced in blastocysts or 2-cell mouse embryos. METHODS: Culture mouse zygotes to two-cell embryos and test effects after 0.5-1 h AMPK agonists' (e.g., Met, BR-DIM) exposure on AMPK-dependent ACCser79P phosphorylation and/or Oct4 by immunofluorescence. Culture morulae to blastocysts and test for increased ACCser79P, decreased Oct4 and for AMPK dependence by coculture with AMPK inhibitor compound C (CC). Test whether Met or BR-DIM decrease growth rates of morulae cultured to blastocyst by counting cells. RESULT(S): Aspirin, metformin, and hyperosmotic sorbitol increased pACC ser79P ~ 20-fold, and BR-DIM caused a ~ 30-fold increase over two-cell embryos cultured for 1 h in KSOMaa but only 3- to 6-fold increase in blastocysts. We previously showed that these stimuli decreased Oct4 40-85% in two-cell embryos that was ~ 60-90% reversible by coculture with AMPK inhibitor CC. However, Oct4 decreased only 30-50% in blastocysts, although reversibility of loss by CC was similar at both embryo stages. Met and BR-DIM previously caused a near-complete cell proliferation arrest in two-cell embryos and here Met caused lower CC-reversible growth decrease and AMPK-independent BR-DIM-induced blastocyst growth decrease. CONCLUSION: Inducing drug or diet supplements decreased anabolism, growth, and stemness have a greater impact on AMPK-dependent processes in two-cell embryos compared to blastocysts.

Commonly used fertility drugs, a diet supplement, and stress force AMPK-dependent block of stemness and development in cultured mammalian embryos.

PURPOSE: The purpose of the present study is to test whether metformin, aspirin, or diet supplement (DS) BioResponse-3,3'-Diindolylmethane (BR-DIM) can induce AMP-activated protein kinase (AMPK)-dependent potency loss in cultured embryos and whether metformin (Met) + Aspirin (Asa) or BR-DIM causes an AMPK-dependent decrease in embryonic development. METHODS: The methods used were as follows: culture post-thaw mouse zygotes to the two-cell embryo stage and test effects after 1-h AMPK agonists' (e.g., Met, Asa, BR-DIM, control hyperosmotic stress) exposure on AMPK-dependent loss of Oct4 and/or Rex1 nuclear potency factors, confirm AMPK dependence by reversing potency loss in two-cell-stage embryos with AMPK inhibitor compound C (CC), test whether Met + Asa (i.e., co-added) or DS BR-DIM decreases development of two-cell to blastocyst stage in an AMPK-dependent (CC-sensitive) manner, and evaluate the level of Rex1 and Oct4 nuclear fluorescence in two-cell-stage embryos and rate of two-cell-stage embryo development to blastocysts. RESULT(S): Met, Asa, BR-DIM, or hyperosmotic sorbitol stress induces rapid ~50-85 % Rex1 and/or Oct4 protein loss in two-cell embryos. This loss is ~60-90 % reversible by co-culture with AMPK inhibitor CC. Embryo development from two-cell to blastocyst stage is decreased in culture with either Met + Asa or BR-DIM, and this is either >90 or ~60 % reversible with CC, respectively. CONCLUSION: These experimental designs here showed that Met-, Asa-, BR-DIM-, or sorbitol stress-induced rapid potency loss in two-cell embryos is AMPK dependent as suggested by inhibition of Rex1 and/or Oct4 protein loss with an AMPK inhibitor. The DS BR-DIM or fertility drugs (e.g., Met + Asa) that are used to enhance maternal metabolism to support fertility can also chronically slow embryo growth and block development in an AMPK-dependent manner.