Quantifying Gut Microbial Short-Chain Fatty Acids and Their Isotopomers in Mechanistic Studies Using a Rapid, Readily Expandable LC-MS Platform.

Short-chain fatty acids (SCFAs) comprise the largest group of gut microbial fermentation products. While absorption of most nutrients occurs in the small intestine, indigestible dietary components, such as fiber, reach the colon and are processed by the gut microbiome to produce a wide array of metabolites that influence host physiology. Numerous studies have implicated SCFAs as key modulators of host health, such as in regulating irritable bowel syndrome (IBS). However, robust methods are still required for their detection and quantitation to meet the demands of biological studies probing the complex interplay of the gut-host-health paradigm. In this study, a sensitive, rapid-throughput, and readily expandible UHPLC-QqQ-MS platform using 2-PA derivatization was developed for the quantitation of gut-microbially derived SCFAs, related metabolites, and isotopically labeled homologues. The utility of this platform was then demonstrated by investigating the production of SCFAs in cecal contents from mice feeding studies, human fecal bioreactors, and fecal/bacterial fermentations of isotopically labeled dietary carbohydrates. Overall, the workflow proposed in this study serves as an invaluable tool for the rapidly expanding gut-microbiome and precision nutrition research field.

A multi-glycomic platform for the analysis of food carbohydrates.

Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan-microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health.

Visualization of AP-1 NF-kappaB ternary complexes in living cells by using a BiFC-based FRET.

Protein-protein interactions are essential for maintaining cell structure and for executing almost all cellular processes. Determination of where and how each protein interacts with its partners provides significant insight into proteins' cellular roles. Although several assays, such as FRET and bimolecular fluorescence complementation (BiFC), have been developed and widely used for visualization and identification of protein interactions in living cells, there is no simple and convenient assay to visualize and identify multiple protein complexes in living cells. Because many signaling molecules often function as ternary complexes, availability of an assay for visualization and identification of ternary complexes will significantly expand the repertoire of protein interaction studies in living cells. By using the Fos-Jun-nuclear factor of activated T cells (NFAT) ternary complex as a model and the fluorescent proteins Cerulean and Venus, two mutant proteins of CFP and YFP with better folding and less environment sensitivity, as a donor and acceptor, respectively, we have combined a Venus-based BiFC system with Cerulean to develop a BiFC-based FRET (BiFC-FRET) assay for visualization of ternary complexes in living cells with a conventional three-filter FRET setup. We also have applied the BiFC-FRET to identify a ternary complex formed between Fos-Jun heterodimers and the NF-kappaB subunit, p65. This finding reveals a cross-talk between AP-1 and NF-kappaB. Thus, the BiFC-FRET represents a convenient assay for identification and visualization of ternary complexes in living cells.

The AKT inhibitor triciribine in combination with paclitaxel has order-specific efficacy against Zfp217-induced breast cancer chemoresistance.

We previously identified the transcription factor ZNF217 (human) / Zfp217 (mouse) as an oncogene and prognostic indicator of reduced survival, increased metastasis, and reduced response to therapy in breast cancer patients. Here we investigated the role of Zfp217 in chemotherapy resistance. Preclinical animal models of Zfp217 overexpression were treated with a combination therapy of the microtubule inhibitor epothilone B, doxorubicin (Adriamycin), and cyclophosphamide (EAC). Tumors overexpressing Zfp217 increased their tumor burden compared to control tumors after treatment and accumulated a mammary gland progenitor cell population (K8+K14+). To overcome this chemoresistance after ZNF217 overexpression, we treated tumors ± Zfp217 overexpression with paclitaxel and triciribine, a nucleoside analog and AKT inhibitor that kills cells that overexpress ZNF217. Treatment order critically impacted the efficacy of the therapy. Combination treatment of triciribine followed by paclitaxel (TCN→PAC) inhibited tumor burden and increased survival in tumors that overexpressed Zfp217, whereas single agent or combination treatment in the reverse order (PAC→TCN) did not improve response. Analysis of these tumors and patient-derived tumor xenograft tumors treated with the same therapies suggested that Zfp217 overexpression in tumors contributes both to decreased microvessel density and vessel maturity, while TCN→PAC tumors overexpressing Zfp217 showed improved vessel maturity.

Patient-Derived Tumor Xenograft Models of Breast Cancer.

The need for model systems that more accurately predict patient outcome has led to a renewed interest and a rapid development of orthotopic transplantation models designed to grow, expand, and study patient-derived human breast tumor tissue in mice. After implanting a human breast tumor piece into a mouse mammary fat pad and allowing the tumor to grow in vivo, the tumor tissue can be either harvested and immediately implanted into mice or can be stored as tissue pieces in liquid nitrogen for surgical implantation at a later time. Here, we describe the process of surgically implanting patient-derived breast tumor tissue into the mammary gland of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice and harvesting tumor tissue for long-term storage in liquid nitrogen.

Targeting CREB inhibits radiation-induced neuroendocrine differentiation and increases radiation-induced cell death in prostate cancer cells.

Neuroendocrine differentiation (NED) is a process by which prostate cancer cells transdifferentiate into neuroendocrine-like (NE-like) cancer cells. Accumulated evidence suggests that NED is associated with disease progression and therapy resistance in prostate cancer patients. We previously reported that by mimicking a clinical radiotherapy protocol, fractionated ionizing radiation (FIR) induces NED in prostate cancer cells. Interestingly, FIR-induced NED constitutes two distinct phases: a radioresistance phase in which a fraction of cells selectively survive during the first two week irradiation, and a neuroendocrine differentiation phase in which surviving cells differentiate into NE-like cancer cells during the second two week irradiation. We have also observed increased activation of the transcription factor cAMP response element binding (CREB) protein during the course of FIR-induced NED. To determine whether targeting NED can be explored as a radiosensitization approach, we employed two CREB targeting strategies, CREB knockdown and overexpression of ACREB, a dominant-negative mutant of CREB, to target both phases. Our results showed that ACREB expression increased FIR-induced cell death and sensitized prostate cancer cells to radiation. Consistent with this, knockdown of CREB also inhibited FIR-induced NED and sensitized prostate cancer cells to radiation. Molecular analysis suggests that CREB targeting primarily increases radiation-induced pre-mitotic apoptosis. Taken together, our results suggest that targeting NED could be developed as a radiosensitization approach for prostate cancer radiotherapy.

Visualization of ternary complexes in living cells by using a BiFC-based FRET assay.

Studies of protein interactions have increased our understanding and knowledge of biological processes. Assays that utilize fluorescent proteins, such as fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), have enabled direct visualization of protein interactions in living cells. However, these assays are primarily suitable for a pair of interacting proteins, and methods to visualize and identify multiple protein complexes in vivo are very limited. This protocol describes the recently developed BiFC-FRET assay, which allows visualization of ternary complexes in living cells. We discuss how to design the BiFC-FRET assay on the basis of the validation of BiFC and FRET assays and how to perform transfection experiments for acquisition of fluorescent images for net FRET calculation. We also provide three methods for normalization of the FRET efficiency. The assay employs a two-chromophore and three-filter FRET setup and is applicable to epifluorescence microscopes. The entire protocol takes about 2-3 weeks to complete.