22 research outputs found
Bio-inspired Composite Hydrogels for Osteochondral Regenerative Engineering
Treatment of osteochondral defects encompassing injury or degeneration to both the articular cartilage as well as the underlying subchondral bone presents a significant medical challenge. Current treatment options including autografts and allografts suffer from limited availability and risk of immunogenicity, respectively. The long term goal of this work is to develop an integrated scaffold system for treatment of osteochondral defects via in situ regeneration of bone, cartilage and the bone-cartilage interface. Hydrogels composed of polymer networks swollen in water provide an attractive biomaterial platform for regeneration of cartilage. In the present study, we have developed a novel composite hydrogel consisting of thiolated hyaluronic acid (HA) and chondroitin sulfate (CS) crosslinked with polyethylene glycol (PEG). The combination of HA and CS offers a biomimetic microenvironment found in cartilage whereas the selection of PEG as a crosslinker is based on its established biocompatibility and chemical versatility. Variations in the crosslinking density enable the ability to fine-tune physical properties of hydrogels. For example, the rheology tests of different hydrogels with increased crosslinking densities showed an increase in equilibrium gel modulus. In vitro study with human mesenchymal stem cells (hMSCs) demonstrated the ability of the hydrogel to support three-dimensional cell encapsulation with high viability. Interestingly, increased crosslinking also promoted phosphorylation of focal adhesion kinase, a potential early mechanosensor that respond to changes in mechanical stiffness. Future in vitro and in vivo studies will be performed to optimize the hydrogels for chondrogenic cellular responses and osteochondral regeneration
Monitoring focal adhesion kinase phosphorylation dynamics in live cells
Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase essential for a diverse set of cellular functions. Current methods for monitoring FAK activity in response to an extracellular stimulus lack spatiotemporal resolution and/or the ability to perform multiplex detection. Here we report on a novel approach to monitor the real-time kinase phosphorylation activity of FAK in live single cells by fluorescence lifetime imaging
Therapeutic Targeting of TFE3/IRS-1/PI3K/mTOR Axis in Translocation Renal Cell Carcinoma
Purpose: Translocation renal cell carcinoma (tRCC) represents a rare subtype of kidney cancer associated with various TFE3, TFEB, or MITF gene fusions that are not responsive to standard treatments for RCC. Therefore, the identification of new therapeutic targets represents an unmet need for this disease.
Experimental Design: We have established and characterized a tRCC patient-derived xenograft, RP-R07, as a novel preclinical model for drug development by using next-generation sequencing and bioinformatics analysis. We then assessed the therapeutic potential of inhibiting the identified pathway using in vitro and in vivo models.
Results: The presence of a SFPQ-TFE3 fusion [t(X;1) (p11.2; p34)] with chromosomal break-points was identified by RNA-seq and validated by RT-PCR. TFE3 chromatin immunoprecipitation followed by deep sequencing analysis indicated a strong enrichment for the PI3K/AKT/mTOR pathway. Consistently, miRNA microarray analysis also identified PI3K/AKT/mTOR as a highly enriched pathway in RP-R07. Upregulation of PI3/AKT/mTOR pathway in additional TFE3âtRCC models was confirmed by significantly higher expression of phospho-S6 (P < 0.0001) and phospho-4EBP1 (P < 0.0001) in established tRCC cell lines compared with clear cell RCC cells. Simultaneous vertical targeting of both PI3K/AKT and mTOR axis provided a greater antiproliferative effect both in vitro (P < 0.0001) and in vivo (P < 0.01) compared with single-node inhibition. Knockdown of TFE3 in RP-R07 resulted in decreased expression of IRS-1 and inhibited cell proliferation.
Conclusions: These results identify TFE3/IRS-1/PI3K/AKT/mTOR as a potential dysregulated pathway in TFE3âtRCC, and suggest a therapeutic potential of vertical inhibition of this axis by using a dual PI3K/mTOR inhibitor for patients with TFE3âtRCC
EZH2 modifies sunitinib resistance in renal cell carcinoma by kinome reprogramming
Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represent a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft (PDX) model that is intrinsically resistant to the RTKI sunitinib but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its anti-angiogenic and anti-metastatic activity but lost its direct anti-tumor effects due to kinome reprogramming, which resulted in suppression of pro- apoptotic and cell cycle regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTK, restoring the anti-tumor effects of sunitnib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease.This research was funded by Roswell Park Cancer Instituteâs Cancer Center Support Grant from National Cancer Institute, NIH P30CA016056 (RP) and a generous donation by Richard and Deidre Turner (RP). This investigation was conducted in-part in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 RR020128-01 from the National Center for Research Resources, National Institutes of Health
Diversity of two forms of DNA methylation in the brain
DNA methylation 5-methylcytosine (5mC) predicts a compacting chromatin inaccessible to transcription. The discovery of 5-hydroxymethylcytosine (5hmC), which is derived from 5mC, adds a new dimension to the mechanism and role of DNA methylation in epigenetics. Genomic evidence indicates that the 5hmC is located in the alternate regions to 5mC. However, the nature of 5hmC, as compared with classical 5mC remains unclear. Observing the mouse brain through embryonic development to the adult, first, we found that 5hmC is not merely an intermediate metabolite of demethylation, but is long lasting, chromatically distinct, and dynamically changing during neurodevelopment. Second, we found that 5hmC distinctly differs from 5mC in its chromatin affiliation during neural stem cell (NSC) development. Thirdly, we found both 5mC and 5hmC to be uniquely polarized and dynamic through the NSC development. 5mC was found to progressively polarize with MBD1 and MeCP2, and recruits H3K9me3 and H3K27me3; while 5hmC progressively co-localizes with MBD3 and recruits H3K4me2. Critical differential binding of 5mC with MBD1, and 5hmC with MBD3 was validated by Resonance Energy Transfer technique FLIM-FRET. This transition and polarization coincides with neuroprogenitor differentiation. Finally, at the time of synaptogenesis, 5mC gradually accumulates in the heterochromatin while 5hmC accumulates in the euchromatin, which is consistent with the co-localization of 5hmC with PolII, which mediates RNA transcription. Our data indicate that 5mC and 5hmC are diverse in their functional interactions with chromatin. This diversity is likely to contribute to the versatile epigenetic control of transcription mediating brain development and functional maintenance of adult brain
Differentiation of cancer cells in two-dimensional and three-dimensional breast cancer models by Raman spectroscopy
Mitochondrial Dysfunction, Disruption of FâActin Polymerization, and Transcriptomic Alterations in Zebrafish Larvae Exposed to Trichloroethylene
Trichloroethylene
(TCE) is primarily used as an industrial degreasing
agent and has been in use since the 1940s. TCE is released into the
soil, surface, and groundwater. From an environmental and regulatory
standpoint, more than half of Superfund hazardous waste sites on the
National Priority List are contaminated with TCE. Occupational exposure
to TCE occurs primarily via inhalation, while environmental TCE exposure
also occurs through ingestion of contaminated drinking water. Current
literature links TCE exposure to various adverse health effects including
cardiovascular toxicity. Current studies aiming to address developmental
cardiovascular toxicity utilized rodent and avian models, with the
majority of studies using relatively higher parts per million (mg/L)
doses. In this study, to further investigate developmental cardiotoxicity
of TCE, zebrafish embryos were treated with 0, 10, 100, or 500 parts
per billion (ppb; ÎŒg/L) TCE during embryogenesis and/or through
early larval stages. After the appropriate exposure period, angiogenesis,
F-actin, and mitochondrial function were assessed. A significant doseâresponse
decrease in angiogenesis, F-actin, and mitochondrial function was
observed. To further complement this data, a transcriptomic profile
of zebrafish larvae was completed to identify gene alterations associated
with the 10 ppb TCE exposure. Results from the transcriptomic data
revealed that embryonic TCE exposure caused significant changes in
genes associated with cardiovascular disease, cancer, and organismal
injury and abnormalities with a number of targets in the FAK signaling
pathway. Overall, results from our study support TCE as a developmental
cardiovascular toxicant, provide molecular targets and pathways for
investigation in future studies, and indicate a need for continued
priority for environmental regulation