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1.
Biotechnol Bioeng ; 112(3): 438-46, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25220691

ABSTRACT

Directed evolution is a powerful method for engineering proteins towards user-defined goals and has been used to generate novel proteins for industrial processes, biological research and drug discovery. Typical directed evolution techniques include cellular display, phage display, ribosome display and water-in-oil compartmentalization, all of which physically link individual members of diverse gene libraries to their translated proteins. This allows the screening or selection for a desired protein function and subsequent isolation of the encoding gene from diverse populations. For biotechnological and industrial applications there is a need to engineer proteins that are functional under conditions that are not compatible with these techniques, such as high temperatures and harsh detergents. Cellular High-throughput Encapsulation Solubilization and Screening (CHESS), is a directed evolution method originally developed to engineer detergent-stable G proteins-coupled receptors (GPCRs) for structural biology. With CHESS, library-transformed bacterial cells are encapsulated in detergent-resistant polymers to form capsules, which serve to contain mutant genes and their encoded proteins upon detergent mediated solubilization of cell membranes. Populations of capsules can be screened like single cells to enable rapid isolation of genes encoding detergent-stable protein mutants. To demonstrate the general applicability of CHESS to other proteins, we have characterized the stability and permeability of CHESS microcapsules and employed CHESS to generate thermostable, sodium dodecyl sulfate (SDS) resistant green fluorescent protein (GFP) mutants, the first soluble proteins to be engineered using CHESS.


Subject(s)
Directed Molecular Evolution/methods , Protein Engineering/methods , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Green Fluorescent Proteins , Hydrogen-Ion Concentration , Protein Stability , Recombinant Proteins/metabolism , Sodium Dodecyl Sulfate , Solubility , Temperature
2.
Oncogene ; 34(1): 63-72, 2015 Jan 02.
Article in English | MEDLINE | ID: mdl-24336327

ABSTRACT

Aggressive cancers and embryonic stem (ES) cells share a common gene expression signature. Identifying the key factors/pathway(s) within this ES signature responsible for the aggressiveness of cancers can lead to a potential cure. In this study, we find that SALL4, a gene involved in the maintenance of ES cell self-renewal, is aberrantly expressed in 47.7% of primary human endometrial cancer samples. It is not expressed in normal or hyperplastic endometrial. More importantly, SALL4 expression is positively correlated with worse patient survival and aggressive features such as metastasis in endometrial carcinoma. Further functional studies have shown that loss of SALL4 inhibits endometrial cancer cell growth in vitro and tumorigenicity in vivo, as a result of inhibition of cell proliferation and increased apoptosis. In addition, downregulation of SALL4 significantly impedes the migration and invasion properties of endometrial cancer cells in vitro and their metastatic potential in vivo. Furthermore, manipulation of SALL4 expression can affect drug sensitivity of endometrial cancer cells to carboplatin. Moreover, we show that SALL4 specifically binds to the c-Myc promoter region in endometrial cancer cells. While downregulation of SALL4 leads to a decreased expression of c-Myc at both protein and mRNA levels, ectopic SALL4 overexpression causes increased c-Myc protein and mRNA expression, indicating that c-Myc is one of the SALL4 downstream targets in endometrial tumorigenesis. In summary, we are the first to demonstrate that SALL4 has functional role(s) in metastasis and drug resistance in aggressive endometrial cancer. As a consequence of its functional roles in cancer cell and absence in normal tissue, SALL4 is a potential novel therapeutic target for the high-risk endometrial cancer patient population.


Subject(s)
Embryonic Stem Cells/metabolism , Endometrial Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Transcription Factors/metabolism , Adult , Aged , Animals , Apoptosis , Carboplatin/pharmacology , Cell Line, Tumor , Cell Proliferation , Drug Resistance, Neoplasm , Female , Gene Silencing , Humans , Immunohistochemistry , Mice , Middle Aged , Neoplasm Metastasis , Neoplasm Transplantation , Proto-Oncogene Proteins c-myc/metabolism , Treatment Outcome , Wound Healing
3.
AJNR Am J Neuroradiol ; 35(2): 402-6, 2014 Feb.
Article in English | MEDLINE | ID: mdl-23907246

ABSTRACT

SUMMARY: Bannayan-Riley-Ruvalcaba syndrome is a congenital disorder characterized by macrocephaly, intestinal polyposis, lipomas, and pigmented macules of the penis. There is limited published radiologic literature on the syndrome. The purpose of this study was to review the brain MR imaging findings in Bannayan-Riley-Ruvalcaba syndrome as well as to compare and contrast the findings with other brain disorders that also have brain cysts and white matter lesions. All brain MR imaging studies were reviewed in patients with a diagnosis of Bannayan-Riley-Ruvalcaba syndrome from our hospital. All 7 patients were evaluated with brain MR imaging. MR imaging results showed white matter cysts in the parietal lobe (7/7), frontal lobe (3/7), and temporal lobe (1/7). These were predominantly surrounded by white matter T2 hyperintensities associated with macrocephaly. Cystic lesions on MR imaging in Bannayan-Riley-Ruvalcaba syndrome are prevalent, and knowledge of this differential diagnosis can allow the radiologist to suggest a diagnosis of this condition in a child with macrocephaly.


Subject(s)
Brain Diseases/pathology , Cysts/pathology , Hamartoma Syndrome, Multiple/pathology , Magnetic Resonance Imaging/methods , Neuroimaging/methods , Child , Child, Preschool , Female , Humans , Infant , Male , Reproducibility of Results , Sensitivity and Specificity
4.
Br J Cancer ; 108(10): 2013-20, 2013 May 28.
Article in English | MEDLINE | ID: mdl-23632482

ABSTRACT

BACKGROUND: Paclitaxel has recently been reported by this laboratory to potentiate the high-LET radiation therapeutic (212)Pb-TCMC-trastuzumab, which targets HER2. To elucidate mechanisms associated with this therapy, targeted α-particle radiation therapeutic (212)Pb-TCMC-trastuzumab together with paclitaxel was investigated for the treatment of disseminated peritoneal cancers. METHODS: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pre-treated with paclitaxel, followed by treatment with (212)Pb-TCMC-trastuzumab and compared with groups treated with paclitaxel alone, (212)Pb-TCMC-HuIgG, (212)Pb-TCMC-trastuzumab and (212)Pb-TCMC-HuIgG after paclitaxel pre-treatment. RESULTS: (212)Pb-TCMC-trastuzumab with paclitaxel given 24 h earlier induced increased mitotic catastrophe and apoptosis. The combined modality of paclitaxel and (212)Pb-TCMC-trastuzumab markedly reduced DNA content in the S-phase of the cell cycle with a concomitant increase observed in the G2/M-phase. This treatment regimen also diminished phosphorylation of histone H3, accompanied by an increase in multi-micronuclei, or mitotic catastrophe in nuclear profiles and positively stained γH2AX foci. The data suggests, possible effects on the mitotic spindle checkpoint by the paclitaxel and (212)Pb-TCMC-trastuzumab treatment. Consistent with this hypothesis, (212)Pb-TCMC-trastuzumab treatment in response to paclitaxel reduced expression and phosphorylation of BubR1, which is likely attributable to disruption of a functional Aurora B, leading to impairment of the mitotic spindle checkpoint. In addition, the reduction of BubR1 expression may be mediated by the association of a repressive transcription factor, E2F4, on the promoter region of BubR1 gene. CONCLUSION: These findings suggest that the sensitisation to therapy of (212)Pb-TCMC-trastuzumab by paclitaxel may be associated with perturbation of the mitotic spindle checkpoint, leading to increased mitotic catastrophe and cell death.


Subject(s)
Colonic Neoplasms/drug therapy , Colonic Neoplasms/radiotherapy , Lead Radioisotopes/therapeutic use , M Phase Cell Cycle Checkpoints/drug effects , Paclitaxel/therapeutic use , Radioimmunotherapy , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/therapeutic use , Cell Death/drug effects , Cell Line, Tumor , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Combined Modality Therapy , Female , Humans , Mice , Mice, Nude , Paclitaxel/pharmacology , Radioimmunotherapy/methods , Treatment Outcome , Xenograft Model Antitumor Assays
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