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1.
Adv Healthc Mater ; 11(7): e2101263, 2022 04.
Article in English | MEDLINE | ID: mdl-34519442

ABSTRACT

Since the seminal work of Wichterle in 1965 describing the first soft contact lenses and their potential for ocular drug delivery, the field has yet to realize his vision. Maintaining all lens commercial properties combined with a mechanism for controlled drug release of therapeutically relevant concentrations for duration of wear is a major challenge. Here, successful in vivo week-long sustained release of a small molecular weight therapeutic in rabbits from extended-wear silicone hydrogel contact lenses meeting all commercial specifications by utilizing a novel macromolecular memory strategy is reported for the first time. Lens-treated eyes show a continuous, therapeutically relevant bromfenac tear concentration of 256.4 ± 23.1 µg mL-1 for 8 days. Bromday (bromfenac ophthalmic solution, 0.09%, Bausch+Lomb) topical drops exhibit a quick peak concentration of 269.3 ± 85.7 µg mL-1 and 100 min duration. Bioavailability (AUC0-8days ) and mean residence time of lenses are 26 and 155 times higher than drops, respectively. Lenses are safe, well tolerated, and no corneal histological differences are observed. This work highlights the enormous potential of drug releasing lenses as a platform strategy, and offers a new dropless clinical strategy for post-cataract, uveitis, post-LASIK, and corneal abrasion treatment.


Subject(s)
Contact Lenses, Extended-Wear , Contact Lenses, Hydrophilic , Animals , Cornea , Hydrogel, Polyethylene Glycol Dimethacrylate , Hydrogels , Rabbits , Silicones
2.
Molecules ; 22(9)2017 Sep 07.
Article in English | MEDLINE | ID: mdl-28880225

ABSTRACT

This paper reports the synthesis and characterization of silver oxide films for use as bactericidal coatings. Synthesis parameters, dissolution/elution rate, and bactericidal efficacy are reported. Synthesis conditions were developed to create AgO, Ag2O, or mixtures of AgO and Ag2O on surfaces by reactive magnetron sputtering. The coatings demonstrate strong adhesion to many substrate materials and impede the growth of all bacterial strains tested. The coatings are effective in killing Escherichia coli and Staphylococcus aureus, demonstrating a clear zone-of-inhibition against bacteria growing on solid media and the ability to rapidly inhibit bacterial growth in planktonic culture. Additionally, the coatings exhibit very high elution of silver ions under conditions that mimic dynamic fluid flow ranging between 0.003 and 0.07 ppm/min depending on the media conditions. The elution of silver ions from the AgO/Ag2O surfaces was directly impacted by the complexity of the elution media, with a reduction in elution rate when examined in complex cell culture media. Both E. coli and S. aureus were shown to bind ~1 ppm Ag⁺/mL culture. The elution of Ag⁺ resulted in no increases in mammalian cell apoptosis after 24 h exposure compared to control, but apoptotic cells increased to ~35% by 48 and 72 h of exposure. Taken together, the AgO/Ag2O coatings described are effective in eliciting antibacterial activity and have potential for application on a wide variety of surfaces and devices.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Oxides/chemistry , Oxides/pharmacology , Silver Compounds/chemistry , Silver Compounds/pharmacology , Silver/chemistry , 3T3 Cells , Animals , Cell Survival , Escherichia coli/drug effects , Humans , Ions/chemistry , Mice , Microbial Sensitivity Tests/methods , Staphylococcus aureus/drug effects , Surface Properties
3.
Cancer Res ; 74(17): 4741-51, 2014 Sep 01.
Article in English | MEDLINE | ID: mdl-25038227

ABSTRACT

The Notch signaling pathway governs many distinct cellular processes by regulating transcriptional programs. The transcriptional response initiated by Notch is highly cell context dependent, indicating that multiple factors influence Notch target gene selection and activity. However, the mechanism by which Notch drives target gene transcription is not well understood. Herein, we identify and characterize a novel Notch-interacting protein, Notch activation complex kinase (NACK), which acts as a Notch transcriptional coactivator. We show that NACK associates with the Notch transcriptional activation complex on DNA, mediates Notch transcriptional activity, and is required for Notch-mediated tumorigenesis. We demonstrate that Notch1 and NACK are coexpressed during mouse development and that homozygous loss of NACK is embryonic lethal. Finally, we show that NACK is also a Notch target gene, establishing a feed-forward loop. Thus, our data indicate that NACK is a key component of the Notch transcriptional complex and is an essential regulator of Notch-mediated tumorigenesis and development.


Subject(s)
Carcinogenesis/genetics , Receptors, Notch/genetics , Transcriptional Activation/genetics , Animals , Cell Line , Cell Line, Tumor , DNA-Binding Proteins/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Receptor, Notch1/genetics , Signal Transduction/genetics , Transcription, Genetic/genetics
5.
Blood ; 117(10): 2901-9, 2011 Mar 10.
Article in English | MEDLINE | ID: mdl-21217079

ABSTRACT

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic neoplasm characterized by malignant expansion of immature T cells. Activated NOTCH (Notch(IC)) and c-MYC expression are increased in a large percentage of human T-ALL tumors. Furthermore, c-MYC has been shown to be a NOTCH target gene. Although activating mutations of Notch have been found in human T-ALL tumors, there is little evidence that the c-MYC locus is altered in this neoplasm. It was previously demonstrated that Notch and c-Myc-regulated genes have a broadly overlapping profile, including genes involved in cell cycle progression and metabolism. Given that Notch and c-Myc appear to function similarly in T-ALL, we sought to determine whether these two oncogenes could substitute for each other in T-ALL tumors. Here we report that NOTCH(IC) is able to maintain T-ALL tumors formed in the presence of exogenous NOTCH(IC) and c-MYC when exogenous c-MYC expression is extinguished. In contrast, c-MYC is incapable of maintaining these tumors in the absence of NOTCH(IC). We propose that failure of c-MYC to maintain these tumors is the result of p53-mediated apoptosis. These results demonstrate that T-ALL maintenance is dependent on NOTCH(IC), but not c-MYC, demonstrating that NOTCH is oncogenic dominant in T-ALL tumors.


Subject(s)
Genes, myc , Oncogenes/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Receptors, Notch/genetics , Animals , Humans , Mice , Receptors, Notch/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction
6.
Cancer Res ; 70(23): 9991-10001, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-21118961

ABSTRACT

Identifying the functions of proteins, which associate with specific subnuclear structures, is critical to understanding eukaryotic nuclear dynamics. Sp100 is a prototypical protein of ND10/PML nuclear bodies, which colocalizes with Daxx and the proto-oncogenic PML. Sp100 isoforms contain SAND, PHD, Bromo, and HMG domains and are highly sumoylated, all characteristics suggestive of a role in chromatin-mediated gene regulation. A role for Sp100 in oncogenesis has not been defined previously. Using selective Sp100 isoform-knockdown approaches, we show that normal human diploid fibroblasts with reduced Sp100 levels rapidly senesce. Subsequently, small rapidly dividing Sp100 minus cells emerge from the senescing fibroblasts and are found to be highly tumorigenic in nude mice. The derivation of these tumorigenic cells from the parental fibroblasts is confirmed by microsatellite analysis. The small rapidly dividing Sp100 minus cells now also lack ND10/PML bodies, and exhibit genomic instability and p53 cytoplasmic sequestration. They have also activated MYC, RAS, and TERT pathways and express mesenchymal to epithelial transdifferentiation (MET) markers. Reintroduction of expression of only the Sp100A isoform is sufficient to maintain senescence and to inhibit emergence of the highly tumorigenic cells. Global transcriptome studies, quantitative PCR, and protein studies, as well as immunolocalization studies during the course of the transformation, reveal that a transient expression of stem cell markers precedes the malignant transformation. These results identify a role for Sp100 as a tumor suppressor in addition to its role in maintaining ND10/PML bodies and in the epigenetic regulation of gene expression.


Subject(s)
Antigens, Nuclear/genetics , Autoantigens/genetics , Embryonic Stem Cells/metabolism , Fibroblasts/metabolism , Tumor Suppressor Proteins/genetics , Animals , Antigens, Nuclear/metabolism , Autoantigens/metabolism , Blotting, Western , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Cellular Senescence/genetics , Epithelial-Mesenchymal Transition/genetics , Fibroblasts/cytology , Gene Expression Profiling , HEK293 Cells , Humans , Male , Mice , Mice, Nude , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Nuclear Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Promyelocytic Leukemia Protein , Proto-Oncogene Proteins c-myc/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolism , Transplantation, Heterologous , Tumor Suppressor Proteins/metabolism , ras Proteins/metabolism
7.
Cell Cycle ; 9(18): 3667-73, 2010 Sep 15.
Article in English | MEDLINE | ID: mdl-20855943

ABSTRACT

The process of regeneration is most readily studied in species of sponge, hydra, planarian and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a mammalian model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G2/M "arrest," showed that p21(Cip1/Waf1) was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL. p53(-/-) mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21(-/-) mice showed chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cycle-related mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfß/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfß/Smad pathway.


Subject(s)
Regeneration , Tumor Suppressor Protein p53/physiology , Animals , Cell Differentiation , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Ear/injuries , Ear/pathology , Mice , Mice, Knockout , Signal Transduction , Smad Proteins/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Tumor Suppressor Protein p53/metabolism
8.
Cancer Res ; 70(11): 4280-6, 2010 Jun 01.
Article in English | MEDLINE | ID: mdl-20484026

ABSTRACT

K-ras is the most commonly mutated oncogene in pancreatic cancer and its activation in murine models is sufficient to recapitulate the spectrum of lesions seen in human pancreatic ductal adenocarcinoma (PDAC). Recent studies suggest that Notch receptor signaling becomes reactivated in a subset of PDACs, leading to the hypothesis that Notch1 functions as an oncogene in this setting. To determine whether Notch1 is required for K-ras-induced tumorigenesis, we used a mouse model in which an oncogenic allele of K-ras is activated and Notch1 is deleted simultaneously in the pancreas. Unexpectedly, the loss of Notch1 in this model resulted in increased tumor incidence and progression, implying that Notch1 can function as a tumor suppressor gene in PDAC.


Subject(s)
Carcinoma, Pancreatic Ductal/genetics , Genes, Tumor Suppressor , Genes, ras , Pancreatic Neoplasms/genetics , Receptor, Notch1/genetics , Animals , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Disease Progression , Gene Deletion , Gene Expression Regulation, Neoplastic , Mice , Mice, Transgenic , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Receptor, Notch1/deficiency , Receptor, Notch1/metabolism , Signal Transduction , beta Catenin/metabolism
9.
Blood ; 113(8): 1689-98, 2009 Feb 19.
Article in English | MEDLINE | ID: mdl-19001083

ABSTRACT

Notch signaling plays a role in normal lymphocyte development and function. Activating Notch1-mutations, leading to aberrant downstream signaling, have been identified in human T-cell acute lymphoblastic leukemia (T-ALL). While this highlights the contribution of Notch signaling to T-ALL pathogenesis, the mechanisms by which Notch regulates proliferation and survival in normal and leukemic T cells are not fully understood. Our findings identify a role for Notch signaling in G(1)-S progression of cell cycle in T cells. Here we show that expression of the G(1) proteins, cyclin D3, CDK4, and CDK6, is Notch-dependent both in vitro and in vivo, and we outline a possible mechanism for the regulated expression of cyclin D3 in activated T cells via CSL (CBF-1, mammals; suppressor of hairless, Drosophila melanogaster; Lag-1, Caenorhabditis elegans), as well as a noncanonical Notch signaling pathway. While cyclin D3 expression contributes to cell-cycle progression in Notch-dependent human T-ALL cell lines, ectopic expression of CDK4 or CDK6 together with cyclin D3 shows partial rescue from gamma-secretase inhibitor (GSI)-induced G(1) arrest in these cell lines. Importantly, cyclin D3 and CDK4 are highly overexpressed in Notch-dependent T-cell lymphomas, justifying the combined use of cell-cycle inhibitors and GSI in treating human T-cell malignancies.


Subject(s)
CD4-Positive T-Lymphocytes/enzymology , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/metabolism , Cyclins/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Receptor, Notch1/metabolism , Animals , CD4-Positive T-Lymphocytes/pathology , Cell Line, Tumor , Cyclin D3 , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 6/genetics , G1 Phase/physiology , Gene Expression Regulation, Leukemic , Gene Expression Regulation, Neoplastic , Humans , Lymphoma, T-Cell/enzymology , Lymphoma, T-Cell/pathology , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Phosphorylation/physiology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Promoter Regions, Genetic/physiology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Receptor, Notch1/genetics , Receptor, Notch2/genetics , Receptor, Notch2/metabolism , Receptor, Notch3 , Receptor, Notch4 , Receptors, Notch/genetics , Receptors, Notch/metabolism , Retinoblastoma Protein/metabolism , S Phase/physiology , Signal Transduction/physiology
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