Ribosomal Profiling by Gradient Fractionation of Cell Lysates.

Ribosomal profiling is a widely used technique for deep sequencing of ribosome-protected mRNA and for measuring ribosome status in cells. It is a powerful method that is typically employed for monitoring and measuring protein translation status and ribosome activity. Also, it has been used for monitoring the ribosomal stress-responsive events in the ribosome activity. Furthermore, this approach enables understanding of translational regulation, which is invisible in most proteomic approaches. Moreover, this method is known as an important approach for biological discovery such as identification of translation products. Hence, this methodology will be useful for studying cellular events engaging in ribosome assembly, ribosome biogenesis, ribosome activity, translation during the cell cycle, cell proliferation, and growth as well as the ribosomal stress response in mammalian cells.

Nanoparticle encapsulation of non-genotoxic p53 activator Inauhzin-C for improved therapeutic efficacy.

The tumor suppressor protein p53 remains in a wild type but inactive form in ~50% of all human cancers. Thus, activating it becomes an attractive approach for targeted cancer therapies. In this regard, our lab has previously discovered a small molecule, Inauhzin (INZ), as a potent p53 activator with no genotoxicity. Method: To improve its efficacy and bioavailability, here we employed nanoparticle encapsulation, making INZ-C, an analog of INZ, to nanoparticle-encapsulated INZ-C (n-INZ-C). Results: This approach significantly improved p53 activation and inhibition of lung and colorectal cancer cell growth by n-INZ-C in vitro and in vivo while it displayed a minimal effect on normal human Wi38 and mouse MEF cells. The improved activity was further corroborated with the enhanced cellular uptake observed in cancer cells and minimal cellular uptake observed in normal cells. In vivo pharmacokinetic evaluation of these nanoparticles showed that the nanoparticle encapsulation prolongates the half-life of INZ-C from 2.5 h to 5 h in mice. Conclusions: These results demonstrate that we have established a nanoparticle system that could enhance the bioavailability and efficacy of INZ-C as a potential anti-cancer therapeutic.

Comparison of Chemotherapeutic Activities of Rhodamine-Based GUMBOS and NanoGUMBOS.

Rhodamine derivatives have been widely investigated for their mitochondrial targeting and chemotherapeutic properties that result from their lipophilic cationic structures. In previous research, we have found that conversion of Rhodamine 6G into nanoGUMBOS, i.e., nanomaterials derived from a group of uniform materials based on organic salts (GUMBOS), led to selective chemotherapeutic toxicity for cancer cells over normal cells. Herein, we investigate the chemotherapeutic activity of GUMBOS derived from four different rhodamine derivatives, two bearing an ester group, i.e., Rhodamine 123 (R123) and SNAFR-5, and two bearing a carboxylic acid group, i.e., rhodamine 110 (R110) and rhodamine B (RB). In this study, we evaluate (1) relative hydrophobicity via octanol-water partition coefficients, (2) cytotoxicity, and (3) cellular uptake in order to evaluate possible structure-activity relationships between these different compounds. Intriguingly, we found that while GUMBOS derived from R123 and SNAFR-5 formed nanoGUMBOS in aqueous medium, no distinct nanoparticles are observed for RB and R110 GUMBOS. Further investigation revealed that the relatively high water solubility of R110 and RB GUMBOS hinders nanoparticle formation. Subsequently, while R123 and SNAFR-5 displayed selective chemotherapeutic toxicity similar to that of previously investigated R6G nanoGUMBOS, the R110 and RB GUMBOS were lacking in this property. Additionally, the chemotherapeutic toxicities of R123 and SNAFR-5 nanoGUMBOS were also significantly greater than R110 and RB GUMBOS. Observed results were consistent with decreased cellular uptake of R110 and RB as compared to R123 and SNAFR-5 compounds. Moreover, these results are also consistent with previous observations that suggest that nanoparticle formation is critical to the observed selective chemotherapeutic properties as well as the chemotherapeutic efficacy of rhodamine nanoGUMBOS.

Crotonylation at serine 46 impairs p53 activity.

Post-translational modifications (PTMs) play pivotal roles in controlling the stability and activity of the tumor suppressor p53 in response to distinct stressors. Here we report an unexpected finding of a short chain fatty acid modification of p53 in human cells. Crotonic acid (CA) treatment induces p53 crotonylation, but surprisingly reduces its protein, but not mRNA level, leading to inhibition of p53 activity in a dose dependent fashion. Surprisingly this crotonylation targets serine 46, instead of any predicted lysine residues, of p53, as detected in TCEP-probe labeled crotonylation and anti-crotonylated peptide antibody reaction assays. This is further confirmed by substitution of serine 46 with alanine, which abolishes p53 crotonylation in vitro and in cells. CA increases p53-dependent glycolytic activity, and augments cancer cell proliferation in response to metabolic or DNA damage stress. Since serine 46 is only found in human p53, our studies unveil an unconventional PTM unique for human p53, impairing its activity in response to CA. Because CA is likely produced by the gut microbiome, our results also predict that this type of PTM might play a role in early human colorectal neoplasia development by negating p53 activity without mutation of this tumor suppressor gene.

Tumor-Targeting NIRF NanoGUMBOS with Cyclodextrin-Enhanced Chemo/Photothermal Antitumor Activities.

The near-infrared fluorescent (NIRF) dye, IR780, is recognized as a promising theranostic agent and has been widely investigated for imaging, chemotherapeutic, and phototherapeutic applications. However, its poor photostability and nonselective toxicities toward both cancer and normal cells limit its biological applications. Herein, we introduce the use of GUMBOS (a group of uniform materials based on organic salts) developed through counter-anion exchange with IR780 and subsequent nanomaterials (nanoGUMBOS) formed by complexation with cyclodextrin (CD) for enhanced chemo/photothermal therapy. Such CD-based nanoGUMBOS display improved aqueous stability, photostability, and photothermal effects relative to traditional IR780. The examination of in vitro cytotoxicity reveals that CD-based nanoGUMBOS are selectively toxic toward cancer cells and exhibit synergistically enhanced cytotoxicity toward cancer cells upon NIR laser irradiation. Additionally, in vivo NIRF imaging demonstrated selective accumulation of these nanoGUMBOS within the tumor site, indicating tumor-targeting properties. Further in vivo therapeutic study of these CD-based nanoGUMBOS suggests excellent chemo/photothermal antitumor effects. Using these studies, we herein demonstrate a promising strategy, via conversion of IR780 into nanoGUMBOS, that can be used for improved theranostic cancer treatment.

Endocytic Selective Toxicity of Rhodamine 6G nanoGUMBOS in Breast Cancer Cells.

Herein, we report on the role of endocytosis in the selective chemotherpeutic toxicity of rhodamine 6G (R6G) based nanomaterials, i.e., nanoGUMBOS, that are derived from a group of uniform materials based on organic salts (GUMBOS). Evaluation of cellular uptake in the presence and absence of endocytosis inhibitors suggests nanoGUMBOS internalization via clathrin-mediated endocytosis in cancer cells and reveals lack of endocytic internalization in normal cells. Results from characterization of these nanomaterials suggest that endocytic internalization in cancer cells leads to nanoGUMBOS dissociation within the endosomal environment. This ultimately results in selective cytotoxicity of the nanoGUMBOS for cancer cells with no toxicity toward normal cells under examined conditions. Following examination of the selectivity mechanism, in vivo investigations were performed to examine potential therapeutic properties of these nanoparticles. Remarkably, nanoGUMBOS treatment using a mouse xenograft model reduced the tumor volume by 50% suggesting retention of in vitro therapeutic properties in vivo. These results corroborate the selective behavior of nanoGUMBOS and demonstrate their in vivo therapeutic effects, providing further insight into the possible use of these nanomaterials as potential chemotherapeutic agents.

Enhanced chemotherapeutic toxicity of cyclodextrin templated size-tunable rhodamine 6G nanoGUMBOS.

Nanodrugs have been widely investigated for combating the large number of side effects associated with conventional therapeutics. Several investigations of such nanomedicines have demonstrated the profound role of nanoparticle size in therapeutic efficacy. Herein, we report the role of cyclodextrin (CD)-templating on the size and therapeutic properties of rhodamine 6G (R6G) nanoGUMBOS, i.e. nanomaterials derived from a Group of Uniform Materials Based on Organic Salts (GUMBOS). In these studies, templating of nanoGUMBOS using 2-hydroxypropyl-alpha (2-HP-α), 2-hydroxypropyl beta (2-HP-ß), and gamma (γ) cyclodextrin (CD) led to a significant reduction in size and enhanced uniformity as indicated by transmission electron microscopy (TEM) images. In addition, CD-templated nanoGUMBOS remarkably displayed a three to four fold enhancement in toxicity towards cancer cells as compared to nanoGUMBOS without CD-templates, suggesting a significant improvement in therapeutic efficacy. Correlation between size and toxicity suggests that CD-templated nanoparticles of ∼70 to 80 nm produced optimal toxicity. Even more interesting, all investigated nanoGUMBOS displayed no toxicity toward normal cells under examined conditions. Moreover, these nanoGUMBOS display comparable chemotherapeutic toxicity to the parent dye, [R6G][Cl], while also eliminating toxicity towards normal cells, indicating their strong chemotherapeutic potential. The studies outlined here provide further insight into an approach that may be employed for rapid synthesis of size tunable nanodrugs for enhanced chemotherapeutic efficacy.

Mitochondria targeting IR780-based nanoGUMBOS for enhanced selective toxicity towards cancer cells.

Herein, a simple counter-ion variation strategy is proposed and demonstrated for design of an array of near infrared IR780-based nanoGUMBOS (nanomaterials from a Group of Uniform Materials Based on Organic Salts) to produce enhanced anticancer activity. These nanomaterials were synthesized by direct nanoengineering of IR780-based GUMBOS using a reprecipitation method, without addition of any other materials. Thus, these novel nanomaterials can serve as carrier-free nanodrugs, providing several distinct advantages over conventional chemotherapeutics. Examination of the size and stability of these nanoGUMBOS indicates formation of approximately 100 nm nanoparticles that are stable under biological conditions. Interestingly, in vitro chemotherapeutic applications of these nanoGUMBOS indicate two to four-fold enhanced toxicity towards breast cancer cells as compared to the parent dye, while still maintaining minimal toxicity towards normal cells. The mechanism of cancer toxicity for these nanoGUMBOS was also examined by a study of their sub-cellular localization as well as using a mitochondrial toxicity assay. Analyses of data from these studies revealed that all nanoGUMBOS primarily accumulate in the mitochondria of cancer cells and produce dysfunction in the mitochondria to induce cell death. Using these studies, we demonstrate tunable properties of IR780-based nanoGUMBOS through simple variation of counter-ions, thus providing a promising strategy for future design of better nanomedicines to be used for cancer therapy.

Tunable GUMBOS-based sensor array for label-free detection and discrimination of proteins.

There is increasing interest in developing new sensor strategies for accurate detection and identification of proteins, primarily due to their significance in various biological processes. In this regard, fluorometric sensors and sensor arrays have been widely explored as facile and inexpensive analytical tools. In this manuscript, we report a sensor array approach, based on a novel group of 6-(p-toluidino)-2-naphthalenesulfonate (TNS)-based organic salts for sensitive and label-free sensing of proteins. In this proof-of-concept study, three proteins (human serum albumin (HSA), α-antitrypsin and ß-lactoglobulin (ß-lac)) and binary mixtures of HSA and α-antitrypsin were used to evaluate the senor array performance. The fingerprint sensor-response patterns, dependent on proteins and protein mixtures at different concentrations, generated from the TNS-based sensors were employed to develop predictive models using principal component analysis (PCA) and linear discriminant analysis (LDA). Interestingly, there is excellent correlation between clustering patterns of PCA and different concentrations of proteins and protein mixtures, which was employed for discrimination of proteins and protein mixtures regardless of concentrations. Furthermore, identification accuracy of the proposed fluorescence-sensor technique, towards discrimination of various concentrations of proteins and protein mixtures, was calculated to be 100%. Overall, this sensor strategy is found to be a very promising tool for accurate discrimination of absolute and relative concentrations of proteins and protein mixtures.

Biofuel: an alternative to fossil fuel for alleviating world energy and economic crises.

The time has come when it is desirable to look for alternative energy resources to confront the global energy crisis. Consideration of the increasing environmental problems and the possible crisis of fossil fuel availability at record high prices dictate that some changes will need to occur sooner rather than later. The recent oil spill in the Gulf of Mexico is just another example of the environmental threats that fossil fuels pose. This paper is an attempt to explore various bio-resources such as corn, barley, oat, rice, wheat, sorghum, sugar, safflower, and coniferous and non-coniferous species for the production of biofuels (ethanol and biodiesel). In order to assess the potential production of biofuel, in this paper, countries are organized into three groups based on: (a) geographic areas; (b) economic development; and(c) lending types, as classified by the World Bank. First, the total fossil fuel energy consumption and supply and possible carbon emission from burning fossil fuel is projected for these three groups of countries. Second, the possibility of production of biofuel from grains and vegetative product is projected. Third, a comparison of fossil fuel and biofuel is done to examine energy sustainability issues.