BackgroundQuantitative polymerase chain reactions (qPCR) are used to monitor relative changes in very small amounts of DNA. One drawback to qPCR is reproducibility: measuring the same sample multiple times can yield data that is so noisy that important differences can be dismissed. Numerous analytical methods have been employed that can extract the relative template abundance between samples. However, each method is sensitive to baseline assignment and to the unique shape profiles of individual reactions, which gives rise to increased variance stemming from the analytical procedure itself.Principal FindingsWe developed a simple mathematical model that accurately describes the entire PCR reaction profile using only two reaction variables that depict the maximum capacity of the reaction and feedback inhibition. This model allows quantification that is more accurate than existing methods and takes advantage of the brighter fluorescence signals from later cycles. Because the model describes the entire reaction, the influences of baseline adjustment errors, reaction efficiencies, template abundance, and signal loss per cycle could be formalized. We determined that the common cycle-threshold method of data analysis introduces unnecessary variance because of inappropriate baseline adjustments, a dynamic reaction efficiency, and also a reliance on data with a low signal-to-noise ratio.SignificanceUsing our model, fits to raw data can be used to determine template abundance with high precision, even when the data contains baseline and signal loss defects. This improvement reduces the time and cost associated with qPCR and should be applicable in a variety of academic, clinical, and biotechnological settings.
Targeted protein degradation is a powerful tool that can be used to create unique physiologies depleted of important factors. Current strategies involve modifying a gene of interest such that a degradation peptide is added to an expressed target protein and then conditionally activating proteolysis, either by expressing adapters, unmasking cryptic recognition determinants, or regulating protease affinities using small molecules. For each target, substantial optimization may be required to achieve a practical depletion, in that the target remains present at a normal level prior to induction and is then rapidly depleted to levels low enough to manifest a physiological response. Here, we describe a simplified targeted degradation system that rapidly depletes targets and that can be applied to a wide variety of proteins without optimizing target protease affinities. The depletion of the target is rapid enough that a primary physiological response manifests that is related to the function of the target. Using ribosomal protein S1 as an example, we show that the rapid depletion of this essential translation factor invokes concomitant changes to the levels of several mRNAs, even before appreciable cell division has occurred.T raditional approaches to unravel protein-coding gene function include making knockout or conditional mutant strains for comparative studies. In many cases, obtaining a conditional inactivation mutant is not tractable or there may be concerns that the inactivated protein is defective only in one of several traits. When investigating essential genes, knockout strains can be cultured for biochemical analyses when there is a complementing copy of the gene. By placing an essential gene under the control of a regulated promoter, so-called depletion-by-division experiments can be performed wherein the gene is turned off, and then cell division and normal protein turnover are used to deplete the encoded factor from the cell. Complications arise when the depletion of a factor induces downstream physiological responses that are not directly related to the factor's function, one example being the activation of multiple toxins when translation is inhibited (12,52,56). A solution to this problem is to specifically target the encoded gene product for conditional depletion (29). In doing so, the gene remains in its natural context, and the encoded product functions normally at the appropriate dose prior to its removal.Prior studies have demonstrated the utility of this approach for specific cases by reengineering target proteins such that they contain a peptide degradation signal that is recognized by a processive protease (7,15,20,24,29,53). In early examples, a degradation peptide that has a weakened affinity for the protease was selected, and then an adapter protein that increases the effective local concentration of the target near the protease to increase degradation was conditionally expressed (20,29). In another example, a cryptic degradation signal was used that was liberated by the conditional expressio...
e23163 Background: We previously reported, in breast cancer, the effectiveness of a novel therapeutic peptide called CT20p that displays cancer-selective cytotoxicity. CT20p targets for inhibition a macromolecular complex called Chaperonin-Containing TCP-1 (CCT) that is responsible for folding actin and tubulin and other proteins, like STAT3, into their active forms. CCT is composed of 8 subunits (CCT1-8) that are highly conserved among species. In breast cancer cases, we found that the genes for CCT were frequently amplified (CCT2), upregulating the chaperonin’s activity, which correlated with decreased patient survival. Methods: To determine whether CCT is active in other cancers and thereby a broad therapeutic target, we examined the protein levels of CCT2 in colon, liver, prostate and lung cancer by immunohistochemistry (IHC), using human tissue microarrays (TMAs). Genomic data from The Cancer Genome Atlas (TCGA) was used to support IHC findings. Immunoblot probing of three CCT subunits (CCT2, CCT4, CCT5) was performed to determine their relative expression level in five small cell lung cancer (SCLC) cell lines compared to normal lung cells and these cells tested for susceptibility to killing by CT20p. Results: Results of IHC staining for CCT2 were interpreted on a scale of 1 to 4 (with 4 being the strongest staining). We found that with the exception of colon cancer, all cancers expressed high levels of CCT2 with advancing stage. We identified a significant correlation with cancer progression in both small cell lung carcinoma (SCLC) and squamous cell lung carcinoma (SqCLC). While individual levels of the three probed CCT subunits varied amongst the SCLC cell lines, overall CCT expression was higher in the SCLC cells when compared to normal lung cells and correlated with susceptibility to killing by CT20p. Using the TCGA database, CCT gene alterations were detected in clinical lung cancer cases, with amplification of CCT4 gene in SqCLC cases linked to decreased survival. Conclusions: These results indicate that targeted inhibition of CCT through CT20p treatment is a promising treatment approach for those cancers like SCLC that currently lack effective therapeutics to sustain progression-free survival.
Chemotherapeutic agents within targeted nanoparticles typically trigger cell death not only in tumors cells but also in normal cells that non-specifically uptake these nanoparticles. An alternative approach would be to target the delivery of a therapeutic agent that is toxic to cancer cells while less toxic to non-tumor cells. To accomplish this, a Bax-derived peptide (CT20) was developed as a selective cancer-targeting agent, and a folate-decorated hyperbranched polyester (HBPE) nanoparticle was employed to deliver the peptide to prostate-specific membrane antigen (PSMA) (+) prostate cancer cells. The receptor-targeting capability of folic acid towards PSMA (+) in prostate cancer was examined using a fluorescence activatable probe, folate-S-S-doxorubicin, and the PSMA inhibitor 2-PMPA. Additionally, a folate-decorated HBPE nanoparticle was utilized to targeted-deliver the CT20 peptide to prostate cancer cells in vitro as well as in athymic mice. CT20-encapsulated folate (folate-CT20) nanoparticles were used to determine cell cytotoxicity against PSMA-positive and -negative cells using flow cytometry. Furthermore, cytotoxicity of folate-CT20 nanoparticles towards murine macrophages was evaluated to determine the consequences of non-specific uptake; the FDA-approved doxorubicin was used for comparison. Cell death associated with CT20 was determined through a violet ratiometric membrane asymmetry probe combined with SYTOX AADvanced dead cell stain to evaluate membrane asymmetry and membrane permeability. Folate-CT20 nanoparticles’ tumor-targeting and tumor-killing efficacy was evaluated in athymic mice bearing PSMA (+) tumors and PSMA (-) tumors. Nanoparticles were delivered intravenously through the tail vein and the tumor size was assessed with calipers and ultrasound. Folate-S-S-Doxorubicin was uptaken specifically by PSMA (+) prostate cancer cells showing fluorescence within 12 hours and cell death within 24 hours. Cells that were preincubated with 2-PMPA followed by folate-S-S-Doxorubicin demonstrated no cell-associated fluorescence or cytotoxicity. Similarly, folate-decorated nanoparticles were only internalized by PSMA (+) cells. Cytotoxicity of folate-CT20 nanoparticles towards PSMA (+) cancer cells occurred within 48 hours, while no changes occurred in PSMA (-) cells. When murine macrophages were treated with PSMA-targeting nanocarriers and doxorubicin individually, significant cytotoxicity only occurred in doxorubicin-exposed cells. In vivo, folate-CT20 nanoparticles were capable of preventing further PSMA (+) tumor growth and caused PSMA (+) tumor regression in some cases. Moreover, minimal liver and spleen damage was observed. Herein, we demonstrate that the CT20 peptide is capable of displaying specific lethality towards PSMA (+) prostate cancer cells and that folate-CT20 nanoparticles are promising vehicles to deliver CT20 in vitro and in vivo. Citation Format: Orielyz Flores, Daniel Nierenberg, Ana C. Carr, Rania Bassiouni, Arati Limaye, Santimukul Santra, Charalambos Kaittanis, Annette Khaled, Jesus Manuel Perez. Peptide power: A PSMA-targeted CT20 nanoparticle to fight prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3241. doi:10.1158/1538-7445.AM2017-3241
The primary purpose of this study was to determine whether personal and institutional characteristics of professors at the University Center for Biology, Agronomic and Animal Sciences (CUCBA) in the University of Guadalajara, Mexico could be used to predict their adoption of computers and the Internet for traditional classroom instruction and their potential adoption of distance education for learning and for teaching. Four variables explained 18% of the variance in professors' adoption of computers and the Internet for classroom instruction. Potential adopters of distance education for learning were more likely to choose distance education for learning via the Internet, report that their highest level of education was a bachelor's degree, report their subject matter discipline as veterinary science, have been teaching longer and were less likely to be self-taught computer users than those who were not potential adopters. Predictors of the adoption of distance education for teaching were consistent with those identified for the adoption of distance education for learning.
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