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We recently reported that cyclic thiosulfinates are cysteine selective cross-linkers that avoid the “dead-end” modifications that contribute to other cross-linkers’ toxicity. In this study, we generalize the chemistry of cyclic thiosulfinates to that of thiol selective cross-linking and apply them to the synthesis of hydrogels. Thiol-functionalized four-arm poly(ethylene glycol) and hyaluronic acid monomers were cross-linked with 1,2-dithiane-1-oxide to form disulfide cross-linked hydrogels within seconds. The synthesized hydrogel could be reduced with physiological concentrations of glutathione, which modulated hydrogel mechanical properties and degradation kinetics. Bovine serum albumin protein was successfully encapsulated in hydrogel, and diffusion-mediated release was demonstrated in vitro. Hep G2 cells grew in the presence of preformed hydrogel and during hydrogel synthesis, demonstrating acceptable cytotoxicity. We encapsulated cells within a hydrogel and demonstrated cell growth and recovery up to 10 days, with and without cell adhesion peptides. In summary, we report cyclic thiosulfinates as a novel class of cross-linkers for the facile synthesis of biodegradable hydrogels.

There are many open questions regarding the hydration of solvent-exposed non-polar tracts and pockets in proteins. Although water is predicted to de-wet purely repulsive surfaces and evacuate crevices, the extent of de-wetting is unclear when ubiquitous van der Waals interactions are in play. The structural simplicity of synthetic supramolecular hosts imbues them with considerable potential to address this issue. To this end, here we detail a combination of densimetry and molecular dynamics simulations of three cavitands, coupled with calorimetric studies of their complexes with short-chain carboxylates. Our results reveal the range of wettability possible within the ostensibly identical cavitand pockets—which differ only in the presence and/or position of the methyl groups that encircle the portal to their non-polar pockets. The results demonstrate the ability of macrocycles to template water cavitation within their binding sites and show how the orientation of methyl groups can trigger the drying of non-polar pockets in liquid water, which suggests new avenues to control guest complexation.

The zinc finger transcription factor GATA-1 plays an essential role in megakaryocytic and erythroid terminal maturation. Yet the molecular mechanisms that control GATA-1 function are incompletely understood. Many transcription factors, in addition to binding DNA, make important protein-protein interactions that modulate their activity. Here, we show that GATA-1 participates in stable multiprotein complexes ranging from ~100 kDa to greater than 600 kDa in murine L8057 megakaryoblastic cells. We generated stable L8057 cell lines expressing metabolically biotinylated and FLAG epitope tagged GATA-1, and performed tandem anti-FLAG immunoaffinity and streptavidin affinity purification of GATA-1 associated complexes. Mass spectrometry (LC/MS/MS) of co-purified proteins identified the known GATA-1 associated proteins Friend of GATA-1 (FOG-1), SCL, Ldb1, Runx-1/Cbf-β, SP1 and all components of the NuRD complex. In addition, we reproducibly co-purified zfp148 (also called ZBP-89/BERF-1/BFCOL-1, mtβ), a ubiquitously expressed Kruppel-type zinc finger transcription factor that binds GC-rich DNA elements. Zfp148 also co-purified with GATA-1 in erythroid (MEL) cells. Physical interaction between GATA-1 and zfp148 was confirmed by co-immunoprecipitation experiments using an anti-zfp148 antibody, and their interaction domains mapped to a C-terminal region of zfp148 and the zinc finger domain of GATA-1. Morpholino-mediated knock-down of zfp148 expression in zebrafish embryos ablates thrombocyte (equivalent to mammalian megakaryocyte/platelet) development, but does not significantly affect erythroid development (equivalent to mammalian primitive erythropoiesis at this stage of development). In vitro differentiation of murine ES cells containing homozygous disruption zfp148 by genetrap insertion (zfp148gt/gt) shows essentially normal primitive erythropoiesis, but markedly impaired megakaryocytic and definitive erythropoiesis. Preliminary chimeric studies in adult mice demonstrate a failure of zfp148gt/gt ES cells to contribute to mature erythrocytes. These findings suggest that zfp148 cooperates with GATA-1 to control differentiation of erythroid and megakaryocytic lineages. The differential requirement of zfp148 for definitive, but not primitive, erythropoiesis raises the possibility of a role in developmental hemoglobin switching.

The zinc finger transcription factor GATA-1 plays an essential role in megakaryocytic and erythroid terminal maturation. Yet the molecular mechanisms that control GATA-1 function are incompletely understood. Many transcription factors, in addition to binding DNA, make important protein-protein interactions that modulate their activity. Here, we show that GATA-1 participates in stable multiprotein complexes ranging from ~100 kDa to greater than 600 kDa in murine L8057 megakaryoblastic cells. We generated stable L8057 cell lines expressing metabolically biotinylated and FLAG epitope tagged GATA-1, and performed tandem anti-FLAG immunoaffinity and streptavidin affinity purification of GATA-1 associated complexes. Mass spectrometry (LC/MS/MS) of co-purified proteins identified the known GATA-1 associated proteins Friend of GATA-1 (FOG-1), SCL, Ldb1, Runx-1/Cbf-β, SP1 and all components of the NuRD complex. In addition, we reproducibly co-purified zfp148 (also called ZBP-89/BERF-1/BFCOL-1, mtβ), a ubiquitously expressed Kruppel-type zinc finger transcription factor that binds GC-rich DNA elements. Zfp148 also co-purified with GATA-1 in erythroid (MEL) cells. Physical interaction between GATA-1 and zfp148 was confirmed by co-immunoprecipitation experiments using an anti-zfp148 antibody, and their interaction domains mapped to a C-terminal region of zfp148 and the zinc finger domain of GATA-1. Morpholino-mediated knock-down of zfp148 expression in zebrafish embryos ablates thrombocyte (equivalent to mammalian megakaryocyte/platelet) development, but does not significantly affect erythroid development (equivalent to mammalian primitive erythropoiesis at this stage of development). In vitrodifferentiation of murine ES cells containing homozygous disruption zfp148 by genetrap insertion (zfp148gt/gt) shows essentially normal primitive erythropoiesis, but markedly impaired megakaryocytic and definitive erythropoiesis. Preliminary chimeric studies in adult mice demonstrate a failure of zfp148gt/gtES cells to contribute to mature erythrocytes. These findings suggest that zfp148 cooperates with GATA-1 to control differentiation of erythroid and megakaryocytic lineages. The differential requirement of zfp148 for definitive, but not primitive, erythropoiesis raises the possibility of a role in developmental hemoglobin switching.