Keratin hydrogel carrier system for simultaneous delivery of exogenous growth factors and muscle progenitor cells.

Ideal material characteristics for tissue engineering or regenerative medicine approaches to volumetric muscle loss (VML) include the ability to deliver cells, growth factors, and molecules that support tissue formation from a system with a tunable degradation profile. Two different types of human hair-derived keratins were tested as options to fulfill these VML design requirements: (1) oxidatively extracted keratin (keratose) characterized by a lack of covalent crosslinking between cysteine residues, and (2) reductively extracted keratin (kerateine) characterized by disulfide crosslinks. Human skeletal muscle myoblasts cultured on coatings of both types of keratin had increased numbers of multinucleated cells compared to collagen or Matrigel(TM) and adhesion levels greater than collagen. Rheology showed elastic moduli from 10(2) to 10(5) Pa and viscous moduli from 10(1) to 10(4) Pa depending on gel concentration and keratin type. Kerateine and keratose showed differing rates of degradation due to the presence or absence of disulfide crosslinks, which likely contributed to observed differences in release profiles of several growth factors. In vivo testing in a subcutaneous mouse model showed that keratose hydrogels can be used to deliver mouse muscle progenitor cells and growth factors. Histological assessment showed minimal inflammatory responses and an increase in markers of muscle formation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 864-879, 2016.

Complexes of vesicular stomatitis virus matrix protein with host Rae1 and Nup98 involved in inhibition of host transcription.

Vesicular stomatitis virus (VSV) suppresses antiviral responses in infected cells by inhibiting host gene expression at multiple levels, including transcription, nuclear cytoplasmic transport, and translation. The inhibition of host gene expression is due to the activity of the viral matrix (M) protein. Previous studies have shown that M protein interacts with host proteins Rae1 and Nup98 that have been implicated in regulating nuclear-cytoplasmic transport. However, Rae1 function is not essential for host mRNA transport, raising the question of how interaction of a viral protein with a host protein that is not essential for gene expression causes a global inhibition at multiple levels. We tested the hypothesis that there may be multiple M protein-Rae1 complexes involved in inhibiting host gene expression at multiple levels. Using size exclusion chromatography and sedimentation velocity analysis, it was determined that Rae1 exists in high, intermediate, and low molecular weight complexes. The intermediate molecular weight complexes containing Nup98 interacted most efficiently with M protein. The low molecular weight form also interacted with M protein in cells that overexpress Rae1 or cells in which Nup98 expression was silenced. Silencing Rae1 expression had little if any effect on nuclear accumulation of host mRNA in VSV-infected cells, nor did it affect VSV's ability to inhibit host translation. Instead, silencing Rae1 expression reduced the ability of VSV to inhibit host transcription. M protein interacted efficiently with Rae1-Nup98 complexes associated with the chromatin fraction of host nuclei, consistent with an effect on host transcription. These results support the idea that M protein-Rae1 complexes serve as platforms to promote the interaction of M protein with other factors involved in host transcription. They also support the idea that Rae1-Nup98 complexes play a previously under-appreciated role in regulation of transcription.

hnRNPs Relocalize to the cytoplasm following infection with vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) matrix protein inhibits nuclear-cytoplasmic mRNA transport. The goal of this work is to determine whether VSV inhibits the nuclear-cytoplasmic transport of heterogeneous ribonucleoproteins (hnRNPs), which are thought to serve as mRNA export factors. Confocal microscopy experiments showed that hnRNPA1, hnRNPK, and hnRNPC1/C2, but not hnRNPB1 or lamin A/C, are relocalized to the cytoplasm during VSV infection. We determined whether protein import is inhibited by VSV by transfecting cells with a plasmid encoding enhanced green fluorescent protein (EGFP) tagged with either the M9 nuclear localization sequence (NLS) or the classical NLS. These experiments revealed that both the M9 NLS and the classical NLS are functional during VSV infection. These data suggest that the inhibition of protein import is not responsible for hnRNP relocalization during VSV infection but that hnRNP export is enhanced. We found that hnRNPA1 relocalization was significantly reduced following the silencing of the mRNA export factor Rae1, indicating that Rae1 is necessary for hnRNP export. In order to determine the role of hnRNPA1 in VSV infection, we silenced hnRNPA1 in HeLa cells and assayed three aspects of the viral life cycle: host protein synthesis shutoff concurrent with the onset of viral protein synthesis, replication by plaque assay, and cell killing. We observed that host shutoff and replication are unaffected by the reduction in hnRNPA1 but that the rate of VSV-induced apoptosis is slower in cells that have reduced hnRNPA1. These data suggest that VSV promotes hnRNPA1 relocalization in a Rae1-dependent manner for apoptotic signaling.