Epitranscriptomic Mass Spectrometry.

Every chemical group that is added to any one of the canonical ribonucleotides in a transcript would create a specific RNA modification. Currently, 170+ RNA modifications have been identified. A specific epitranscriptome refers to all the RNA modifications in a given biological system and is considered to play an important role in the regulations of cellular activities. Mass spectrometry-based methods have proven to be the most accurate way to identify RNA modifications and determine the amount of each detectable modification. Relating to the recent development of mapping specific RNA modifications within a transcriptome, the profiling of all RNA modifications can serve as a prescreening tool for mapping and provides support for analyzing the data obtained from mapping. In this chapter, the details for setting up a commonly used mass spectrometry-based method to profile all the RNA modifications in specific epitranscriptomes are described, and the possible options if available are discussed.

Carbon Nanodots Inhibit Oxidized Low Density Lipoprotein-Induced Injury and Monocyte Adhesion to Endothelial Cells Through Scavenging Reactive Oxygen Species.

Oxidized low density lipoprotein (Ox-LDL) is a known biomarker of inflammation and atherosclerosis, a leading cause of death worldwide. As a new class of nanomaterials, carbon nanodots (CNDs) are widely used in bioimaging, diagnostics, and drug delivery. However, there is no current report on how these CNDs affect the cardiovascular system, particularly their potential in mediating endothelial inflammatory dysfunction. This study examined effects of CNDs on Ox-LDL-mediated endothelial dysfunction. CNDs significantly inhibited Ox-LDL-mediated adhesion of monocytes to human microvascular endothelial cells (HMEC-1), in human microvascular endothelial cells (HMEC-1). CNDs significantly inhibited Ox-LDL-mediated adhesion of monocytes to endothelial cells, which is an essential step in the development of atherosclerosis. Further, CNDs significantly inhibited OxLDL-induced expression of interleukin-8 (IL-8), a vital cytokine on monocyte adhesion to the endothelial cells. These results demonstrate CNDs possess anti-inflammatory properties. CNDs also protect cells against Ox-LDL-induced cytotoxicity. Electron paramagnetic resonance (EPR) spectroscopy studies demonstrated direct reactive oxygen species-scavenging by CNDs. This result indicates that the anti-inflammatory properties of CNDs are most likely due to their direct scavenging of reactive oxygen species. Animal studies involving mice did not show any morphological or physical changes between the CNDs and control groups. Our study provides evidence of potential of CNDs in reducing Ox-LDL-mediated inflammation and cytotoxicity in HMEC-1.

Recent Advances in Carbon Nanodots: A Promising Nanomaterial for Biomedical Applications.

Carbon nanodots (CNDs) are an emerging class of nanomaterials and have generated much interest in the field of biomedicine by way of unique properties, such as superior biocompatibility, stability, excellent photoluminescence, simple green synthesis, and easy surface modification. CNDs have been featured in a host of applications, including bioimaging, biosensing, and therapy. In this review, we summarize the latest research progress of CNDs and discuss key advances in our comprehension of CNDs and their potential as biomedical tools. We highlighted the recent developments in the understanding of the functional tailoring of CNDs by modifying dopants and surface molecules, which have yielded a deeper understanding of their antioxidant behavior and mechanisms of action. The increasing amount of in vitro research regarding CNDs has also spawned interest in in vivo practices. Chief among them, we discuss the emergence of research analyzing CNDs as useful therapeutic agents in various disease states. Each subject is debated with reflection on future studies that may further our grasp of CNDs.

Anti-Inflammatory Effect and Cellular Uptake Mechanism of Carbon Nanodots in in Human Microvascular Endothelial Cells.

Cardiovascular disease (CVD) has become an increasingly important topic in the field of medical research due to the steadily increasing rates of mortality caused by this disease. With recent advancements in nanotechnology, a push for new, novel treatments for CVD utilizing these new materials has begun. Carbon Nanodots (CNDs), are a new form of nanoparticles that have been coveted due to the green synthesis method, biocompatibility, fluorescent capabilities and potential anti-antioxidant properties. With much research pouring into CNDs being used as bioimaging and drug delivery tools, few studies have been completed on their anti-inflammatory potential, especially in the cardiovascular system. CVD begins initially by endothelial cell inflammation. The cause of this inflammation can come from many sources; one being tumor necrosis factor (TNF-α), which can not only trigger inflammation but prolong its existence by causing a storm of pro-inflammatory cytokines. This study investigated the ability of CNDs to attenuate TNF-α induced inflammation in human microvascular endothelial cells (HMEC-1). Results show that CNDs at non-cytotoxic concentrations reduce the expression of pro-inflammatory genes, mainly Interleukin-8 (IL-8), and interleukin 1 beta (IL-1ß). The uptake of CNDs by HMEC-1s was examined. Results from the studies involving channel blockers and endocytosis disruptors suggest that uptake takes place by endocytosis. These findings provide insights on the interaction CNDs and endothelial cells undergoing TNF-α induced cellular inflammation.

Modulation of Macrophage Polarization by Carbon Nanodots and Elucidation of Carbon Nanodot Uptake Routes in Macrophages.

Atherosclerosis represents an ever-present global concern, as it is a leading cause of cardiovascular disease and an immense public welfare issue. Macrophages play a key role in the onset of the disease state and are popular targets in vascular research and therapeutic treatment. Carbon nanodots (CNDs) represent a type of carbon-based nanomaterial and have garnered attention in recent years for potential in biomedical applications. This investigation serves as a foremost attempt at characterizing the interplay between macrophages and CNDs. We have employed THP-1 monocyte-derived macrophages as our target cell line representing primary macrophages in the human body. Our results showcase that CNDs are non-toxic at a variety of doses. THP-1 monocytes were differentiated into macrophages by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) and co-treatment with 0.1 mg/mL CNDs. This co-treatment significantly increased the expression of CD 206 and CD 68 (key receptors involved in phagocytosis) and increased the expression of CCL2 (a monocyte chemoattractant and pro-inflammatory cytokine). The phagocytic activity of THP-1 monocyte-derived macrophages co-treated with 0.1 mg/mL CNDs also showed a significant increase. Furthermore, this study also examined potential entrance routes of CNDs into macrophages. We have demonstrated an inhibition in the uptake of CNDs in macrophages treated with nocodazole (microtubule disruptor), N-phenylanthranilic acid (chloride channel blocker), and mercury chloride (aquaporin channel inhibitor). Collectively, this research provides evidence that CNDs cause functional changes in macrophages and indicates a variety of potential entrance routes.

Epitranscriptomic profile of Lactobacillus agilis and its adaptation to growth on inulin.

OBJECTIVE: Ribonucleic acids (RNA) are involved in many cellular functions. In general, RNA is made up by only four different ribonucleotides. The modifications of RNA (epitranscriptome) can greatly enhance the structural diversity of RNA, which in turn support some of the RNA functions. To determine whether the epitranscriptome of a specific probiotic is associated with its adaptation to the source of energy, Lactobacillus agilis (YZ050) was selected as a model and its epitranscriptome was profiled and compared by using mass spectrometry. RESULTS: The L. agilis epitranscriptome (minus rRNA modifications) consists of 17 different RNA modifications. By capturing the L. agilis cells during exponential growth, reproducible profiling was achieved. In a comparative study, the standard source of energy (glucose) in the medium was substituted by a prebiotic inulin, and a downward trend in the L. agilis epitranscriptome was detected. This marks the first report on a system-wide variation of a bacterial epitranscriptome that resulted from adapting to an alternative energy source. No correlation was found between the down-regulated RNA modifications and the expression level of corresponding writer genes. Whereas, the expression level of a specific exonuclease gene, RNase J1, was detected to be higher in cells grown on inulin.

A theoretical perspective of the physical properties of different RNA modifications with respect to RNA duplexes.

Epitranscriptomic variations include >140 different RNA modifications, many of which can serve as disease biomarkers. Owing to the challenges on synthesizing modified RNA oligos, majority of earlier studies on the effects of RNA modifications to RNA duplexes focused on selected individual epitranscriptomic variation. There are also limited development on the computational modeling of RNA duplexes containing a specific epitranscriptomic variation. This study aims to theoretically estimate the physical properties of different modified ribonucleosides and compare their variations with respect to altering the molecular structure of an RNA duplex.

Obtusaquinone: A Cysteine-Modifying Compound That Targets Keap1 for Degradation.

We have previously identified the natural product obtusaquinone (OBT) as a potent antineoplastic agent with promising in vivo activity in glioblastoma and breast cancer through the activation of oxidative stress; however, the molecular properties of this compound remained elusive. We used a multidisciplinary approach comprising medicinal chemistry, quantitative mass spectrometry-based proteomics, functional studies in cancer cells, and pharmacokinetic analysis, as well as mouse xenograft models to develop and validate novel OBT analogs and characterize the molecular mechanism of action of OBT. We show here that OBT binds to cysteine residues with a particular affinity to cysteine-rich Keap1, a member of the CUL3 ubiquitin ligase complex. This binding promotes an overall stress response and results in ubiquitination and proteasomal degradation of Keap1 and downstream activation of the Nrf2 pathway. Using positron emission tomography (PET) imaging with the PET-tracer 2-[18F]fluoro-2-deoxy-d-glucose (FDG), we confirm that OBT is able to penetrate the brain and functionally target brain tumors. Finally, we show that an OBT analog with improved pharmacological properties, including enhanced potency, stability, and solubility, retains the antineoplastic properties in a xenograft mouse model.

Towards a Better Understanding of Computational Models for Predicting DNA Methylation Effects at the Molecular Level.

Human DNA is a very sensitive macromolecule and slight changes in the structure of DNA can have disastrous effects on the organism. When nucleotides are modified, or changed, the resulting DNA sequence can lose its information, if it is part of a gene, or it can become a problem for replication and repair. Human cells can regulate themselves by using a process known as DNA methylation. This methylation is vitally important in cell differentiation and expression of genes. When the methylation is uncontrolled, however, or does not occur in the right place, serious pathophysiological consequences may result. Excess methylation causes changes in the conformation of the DNA double helix. The secondary structure of DNA is highly dependent upon the sequence. Therefore, if the sequence changes slightly the secondary structure can change as well. These slight changes will then cause the doublestranded DNA to be more open and available in some places where large adductions can come in and react with the DNA base pairs. Computer models have been used to simulate a variety of biological processes including protein function and binding, and there is a growing body of evidence that in silico methods can shed light on DNA methylation. Understanding the anomeric effect that contributes to the structural and conformational flexibility of furanose rings through a combination of quantum mechanical and experimental studies is critical for successful molecular dynamic simulations.

Nanospray desorption electrospray ionization mass spectrometry of untreated and treated probiotic Lactobacillus reuteri cells.

Mass spectrometry has proven to be a useful technique for rapid identification of bacterial cells. Among various ionization techniques in mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) has been commonly used for the identification of bacterial cells. Recently, MALDI mass spectrometry has also been utilized to distinguish cellular responses. Ambient ionization techniques do support whole bacterial cell analysis, which include desorption electrospray ionization (DESI). Nanospray DESI (nDESI) is a new variant of DESI, and its application to whole-cell mass spectrometry is limited. In this project, the use of nDESI mass spectrometry to measure probiotic Lactobacillus reuteri (LR) cells is explored. A unique and reproducible mass spectral pattern of untreated LR cells was obtained by using 50% methanol/water as nDESI solvent. The use of nDESI mass spectrometry is further extended to distinguish untreated LR cells from treated LR cells that have been exposed to low pH. These findings demonstrate the feasibility of using nDESI in whole-cell mass spectrometry. Graphical abstract ᅟ.

Measuring ß-Galactosidase Activity in Gram-Positive Bacteria Using a Whole-Cell Assay with MUG as a Fluorescent Reporter.

The use of ß-galactosidase enzyme as a biomarker has the potential to determine activity levels of the microbiome of a variety of organisms due to its common presence in both eukaryotes and prokaryotes. Completing the assay in a whole-cell format facilitates the monitoring of ß-galactosidase activity in its actual cellular environment. This unit describes an optimized fluorescent assay for ß-galactosidase that has enough sensitivity to detect the enzymatic activity despite the thick gram-positive bacterial cellular membrane. The use of a smaller fluorometric substrate, namely 4-methylumbelliferyl ß-D-galactopyranoside (MUG), has facilitated its penetration into the cells as well as its direct detection without any extra steps. This assay provides an improved technique for measuring a well-studied reporter enzyme and offers new avenues for using ß-galactosidase as a biomarker. © 2017 by John Wiley & Sons, Inc.

MicroRNA MultiTool: A Software for Identifying Modified and Unmodified Human microRNA Using Mass Spectrometry.

microRNA (miRNA) are short endogenous non-coding RNA that play a crucial role in post-transcriptional gene regulation and have been implicated in the initiation and progression of 160+ human diseases. Excellent analytical methods have been developed for the measurement of miRNA by mass spectrometry. However, interpretation of mass spectrometric data has been an incapacitating bottleneck in miRNA identification. This study details the development of MicroRNA MultiTool, a software for the identification of miRNA from mass spectrometric data. The software includes capabilities such as miRNA search and mass calculator, modified miRNA mass calculator, and miRNA fragment search. MicroRNA MultiTool bridges the gap between experimental data and identification of miRNA by providing a rapid means of mass spectrometric data interpretation.

Fluorometric cell-based assay for ß-galactosidase activity in probiotic gram-positive bacterial cells - Lactobacillus helveticus.

Although methods for measuring ß-galactosidase activity in intact gram-negative bacterial cells have been reported, the methods may not be applicable to measuring ß-galactosidase activity in gram-positive bacterial cells. This report focuses on the development of a fluorometric cell-based assay for measuring ß-galactosidase activity in gram-positive cells.

High Percentage of Isomeric Human MicroRNA and Their Analytical Challenges.

MicroRNA (miR) are short non-coding RNAs known to post-transcriptionally regulate gene expression, and have been reported as biomarkers for various diseases. miR have also been served as potential drug targets. The identity, functions and detection of a specific miR are determined by its RNA sequence, whose composition is made up of only 4 canonical ribonucleotides. Hence, among over two thousand human miR, their nucleotide compositions are expected to be similar but the extent of similarity has not been reported. In this study, the sequences of mature human miR were downloaded from miRBase, and collated using different tools to determine and compare their nucleotide compositions and sequences. 55% of all human miR were found to be structural isomers. The structural isomers of miR (SimiR) are defined as having the same size and identical nucleotide composition. A number of SimiR were also found to have high sequence similarities. To investigate the extent of SimiR in biological samples, three disease models were chosen, and disease-associated miR were identified from miR2Disease. Among the disease models, as high as 73% of miR were found to be SimiR. This report provides the missing information about human miR and highlights the challenges on the detection of SimiR.

Single nucleotide polymorphisms in type 2 diabetes among Hispanic adults.

In this pilot study, we explore the genetic variation that may relate to type 2 diabetes (T2D) among Hispanic adults. The genotypes of 36 Hispanic adults were analyzed by using the Cardio-Metabochip. The goal is to identify single nucleotide polymorphisms (SNPs) associated to T2D among Hispanic adults. A total of 26 SNPs were identified to be associated with T2D among Hispanic adults. None of these SNPs have been reported for T2D. By using the principle components analysis to analyze the genotype of 26 SNPs in 36 samples, the samples obtained from diabetic patients could be distinguished from the control samples. The findings support genetic involvement in T2D among Hispanic adults.

Rapid differentiation of in vitro cellular responses to toxic chemicals by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Changes in protein expression as a cellular response to chemical exposure have been well established. Current methods for monitoring cellular responses usually require the use of specific reagents and/or labor-intensive procedures. The present study demonstrates the concept of using mass spectral pattern to distinguish different cellular responses. The concept is based on the ability to acquire a unique mass spectral pattern directly from a specific cell culture by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results demonstrate that distinguishable and reproducible spectral patterns can be obtained from different cellular responses.

The footprints of gut microbial-mammalian co-metabolism.

Gut microbiota are associated with essential various biological functions in humans through a "network" of microbial-host co-metabolism to process nutrients and drugs and modulate the activities of multiple pathways in organ systems that are linked to different diseases. The microbiome impacts strongly on the metabolic phenotypes of the host, and hence, metabolic readouts can give insights into functional metagenomic activity. We applied an untargeted mass spectrometry (MS) based metabonomics approach to profile normal Wistar rats exposed to a broad spectrum ß-lactam antibiotic imipenem/cilastatin sodium, at 50 mg/kg/daily for 4 days followed by a 14-day recovery period. In-depth metabolic phenotyping allowed identification of a panel of 202 urinary and 223 fecal metabolites significantly related to end points of a functional metagenome (p < 0.05 in at least one day), many of which have not been previously reported such as oligopeptides and carbohydrates. This study shows extensive gut microbiota modulation of host systemic metabolism involving short-chain fatty acids, tryptophan, tyrosine metabolism, and possibly a compensatory mechanism of indole-melatonin production. Given the integral nature of the mammalian genome and metagenome, this panel of metabolites will provide a new platform for potential therapeutic markers and mechanistic solutions to complex problems commonly encountered in pathology, toxicology, or drug metabolism studies.

Investigation of enzymatic behavior of benzonase/alkaline phosphatase in the digestion of oligonucleotides and DNA by ESI-LC/MS.

We have developed an ion-pairing HPLC-MS method that has sufficient separation power, selectivity, and sensitivity to investigate the enzymatic behavior of benzonase/alkaline phosphatase upon digestion of oligonucleotides and DNA. Mass spectrometry revealed that this enzyme pair can nonspecifically digest oligonucleotides and DNA into fragments ranging from 2 to 10 nucleotides, i.e., sizes suitable for routine mass spectrometric measurements. Trimers, tetramers, and pentamers are the most prominent digested products. This makes benzonase/alkaline phosphatase a promising choice for DNA and DNA adduct related studies that require a nonspecific enzyme. A computer software program developed in-house was critical in automating the processing of mass spectral data. The methodology described here provides a systematic approach for evaluating the behavior of DNA-cleaving enzymes by mass spectrometry.

Capillary isoelectric focusing and affinity capillary electrophoresis approaches for the determination of binding constants for antibodies to the prion protein.

For the development of specific immunological assays, the binding of a specific antibody (Ab) to the target antigen (Ag) has to be relatively strong. In this study, we have utilized affinity capillary electrophoresis (ACE), a form of capillary zone electrophoresis (CZE) to determine the binding constant (Kb) of specific Abs against bovine serum albumin (BSA) and the healthy prion protein (PrPc), in buffer solutions at fixed pHs, approximating in vivo conditions. We have also utilized capillary isoelectric focusing (cIEF) to determine the complexity and recognition of the various isoforms of PrPc Abs towards their Ag, PrPc. Only ACE and CZE have been used to derive Kb values. The selected Abs for the prion protein can recognize both healthy and diseased states of the protein and are commercially available. The Kb values of PrPc Abs appear to be as strong as the anti-BSA (Ab to BSA) and other reported Kb values for proteins of similar size to PrPc. This appears to be one of the few reports on Kb values for any PrPc Abs, and their applications for in vitro immunoassays (e.g., enzyme-linked immunosorbent assays (ELISAs)). Such assays are being used to detect the infectious agent, PrPres, in brain and related matter/tissues.

Base-specific fragmentation of amplified 16S rRNA genes analyzed by mass spectrometry: a tool for rapid bacterial identification.

A rapid approach to the 16S rRNA gene (16S rDNA)-based bacterial identification has been developed that combines uracil-DNA-glycosylase (UDG)-mediated base-specific fragmentation of PCR products with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). 16S rDNA signature sequences were PCR-amplified from both cultured and as-yet-uncultured bacteria in the presence of dUTP instead of dTTP. These PCR products then were immobilized onto a streptavidin-coated solid support to selectively generate either sense or antisense templates. Single-stranded amplicons were subsequently treated with uracil-DNA-glycosylase to generate T-specific abasic sites and fragmented by alkaline treatment. The resulting fragment patterns were analyzed by MALDI-TOF MS. Mass signals of 16S rDNA fragments were compared with patterns calculated from published 16S rDNA sequences. MS of base-specific fragments of amplified 16S rDNA allows reliable discrimination of sequences differing by only one nucleotide. This approach is fast and has the potential for high-throughput identification as required in clinical, pharmaceutical, or environmental microbiology. In contrast to identification by MS of intact whole bacterial cells, this technique allows for the characterization of both cultured and as-yet-uncultured bacteria.