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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.

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-[(18)F]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.

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.

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.

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-1β). 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.

In the absence of high vacuum, the mobility of ions in an electric field is dependent on the charge, mass, shape and size of the ions. To achieve accurate calibration for ion mobility measurements, it is important to select calibrants with comparable physical properties to the molecular ions of interest. The size of an ion is often referred as its collision cross section (CCS). Currently, the information on the CCS values of various types of molecular ions are limited, thus representing a challenge to the calibration of ion mobility spectrometry. In this study, instead of finding a way to improve the calibration of ion mobility measurements, the intrinsic by-products of the conventional matrix-assisted laser desorption/ionization (MALDI) technique, namely MALDI matrix cluster (MAC) ions, are being used as internal references for ion mobility measurements. The standard MALDI matrix and sample preparation method are used. During the MALDI ionization process, MAC ions are generated and co-exist with the molecular ions of interest within the ion source. Our results indicate the MAC ions do possess suitable ion mobility characteristics, thus allowing the MAC ions to serve as internal reference for ion mobility measurements. The MAC ions cannot be used as internal standard or calibrants for ion mobility measurements, because their molecular structures as well as their CCS are unknown. However, the detection of MAC ions can allow us to determine whether the normal operation as well as the expected performance on the sensitivity and resolution of ion mobility spectrometry are achievable and reproducible. The MAC ions can also facilitate the transfer of specific experimental protocols between ion mobility instruments and/or laboratories. No extra materials, equipment or procedure are required for using MAC ions. For the proof of concept, all experimental work in this study was carried out on a traveling wave ion mobility mass spectrometry platform.

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.

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, pharmacokinetic analysis, as well as mouse xenograft models to develop and validate novel OBT analogs and charaterize the molecular mechanism of action of OBT. We here show that OBT and analogs, which have improved pharmacological properties, bind to cysteine residues with 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.

Ion mobility mass spectrometry (IM-MS) can be used to differentiate and identify isobaric ions. To improve IM-MS resolution, the second generation of traveling wave ion mobility (TWIM) technology was launched. There were reports showing ions were heated up by TWIM. With higher ion energy, it could alter the conformation of larger ions or MS/MS experiments. To monitor the energy exchange relating to the TWIM process, the combined use of thermometer ions with unique molecular structure and theoretical calculations to determine the effective temperature of ions had been explored. In this report, the use of a simple experimental approach to estimate the variation on the ion energy that result from changing a TWIM parameter setting is demonstrated. The approach aims to achieve the same percentage of ion dissociation in a collision cell, which is part of the original instrument and located at the exit of TWIM cell. Similar to the traditional MS/MS experiments, the same level of ion dissociation could be achieved by adjusting the electrical potential that was applied to the collision cell. The higher the ion energy after the TWIM separation, the lower the electrical potential was required to achieve the same level of ion dissociation. Together with the information on the number of electrical charge in the selected ion, the difference in the required electrical potentials could be converted into electron volt of ion energy that resulted from changing the TWIM parameter setting. The results showed ion energy could be changed 1–9 eV when the parameter of TWIM was adjusted.

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.

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.

The use of β-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 β-galactosidase activity in its actual cellular environment. This unit describes an optimized fluorescent assay for β-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 β-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 β-galactosidase as a biomarker. © 2017 by John Wiley & Sons, Inc.

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.

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

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.

The increasing interest in microRNA (miRNA) as diagnostic biomarkers or potential drug targets has raised the demand for more accurate miRNA detection. One way to improve the accuracy is by using mass spectrometry (MS) to measure miRNA directly. Matrix-assisted laser desorption/ionization (MALDI) MS stands apart from other MS techniques due to the fact that a MALDI matrix is required for sample preparation. In this study, by exploiting the acidity of MALDI matrix and its mixing with miRNA prior to MS measurements, a simple method to generate RNA sequencing ladders is developed. The method utilizes the MALDI matrix to hydrolyze RNA at high temperature. The resulting sequencing ladders are ready to be measured without any desalting. By using MALDI SpiralTOF MS, the monoisotopic mass of each RNA fragment was measured. The RNA sequence was determined by sequentially comparing nucleotide compositions that were calculated from measured monoisotopic masses. The use of nucleotide compositions to assist the spectral interpretation has the advantages on distinguishing the complementary sequencing ladders, and allows the nucleotide identity at each position to be crosschecked multiple times. Together with the analysis of both complementary sequencing ladders, 100% sequence coverage and sequence accuracy were achieved in a blinded study.

For the analysis of microRNA, a common approach is to first extract microRNA from cellular samples prior to any specific microRNA detection. Thus, it is important to determine the quality and yield of extracted microRNA. In this study, solid-phase extraction was used to isolate small RNA (? Green II staining. Testing for contamination of any small DNA fragments, RNase and cellular peptides or proteins were systematically carried out. By scanning the gel image obtained from PAGE analysis, the average percentage of total microRNA (19 - 25 nt) in the extracted RNA samples was determined to be equal to 2.3 ± 0.5%. The yield of total microRNA was calculated to be ~0.5ng of microRNA per milligram of frozen mouse brain tissue. In comparison to other methods that require the use of expensive specialized instrumentation, the approach of combining the standard UV absorbance and PAGE analysis represents a simple and viable method for evaluating the quality and yield of microRNA extraction from tissue samples.

The advantages of combining qualitative and quantitative analysis on a single analytical technique have further extended the applications of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to the quantitation of various biomolecules. To achieve absolute quantitation, it is necessary to perform a calibration with standard dilutions. For the purpose of measuring DNA samples, a pure DNA oligonucleotide at different concentrations was chosen as a standard to perform the calibration of Quantitative MALDI-TOF MS. In order to overcome the variation of signal intensity from repeated measurements of each DNA standard dilution, fixed amount of an internal standard was added into each DNA standard dilution. Instead of maintaining at a constant level, the signals of fixed amount of internal standard were decreased 73% from its initial level while the signals of DNA standard continued to increase within a linear dynamic range for quantitation from 0.20 μM to 12.5 μM of DNA. Attempts to identify the cause of signal reduction were systematically carried out. This is the first report on the extent of signal reduction in quantitative MALDI-TOF MS. These results represent a limitation on using MALDI-TOF MS to monitor the changes in concentration of two different compounds within a chemical or biological system.

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 β-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.

Although the UV absorbance of DNA at 260 nm has been recognized as a standard method for DNA quantitation, there are limitations of using UV spectrometry to determine the purity and identity of DNA. Recently, MALDI-TOF MS has proven to be an accurate technique for qualitative DNA analysis. In this study, the accuracy of MALDI-TOF MS for determining the concentration of DNA is evaluated and compared with that of the standard UV method. The results indicated that the accuracy of quantitative MALDI-TOF MS was comparable to that of the standard UV method and that measured DNA concentrations correlated well with those determined by the standard UV method.

Many chemical carcinogens and their in vivo activated metabolites react readily with genomic DNA, and form covalently bound carcinogen-DNA adducts. Clinically, carcinogen-DNA adducts have been linked to various cancer diseases. Among the current methods for DNA adduct analysis, mass spectroscopic method allows the direct measurement of unlabeled DNA adducts. The goal of this study is to explore the use of matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to determine the identity of carcinogen-DNA adducts. Two of the known carcinogenic DNA adducts, namely N-(2'-deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenyl-imidazo [4,5-b] pyridine (dG-C8-PhIP) and N-(2'-deoxyguanosin-8yl)-4-aminobiphenyl (dG-C8-ABP), were selected as our models. In MALDI-TOF MS measurements, the small matrix ion and its cluster ions did not interfere with the measurements of both selected dG adducts. To achieve a higher accuracy for the characterization of selected dG adducts, 1 keV collision energy in MALDI-TOF/TOF MS/MS was used to measure the adducts. In comparison to other MS/MS techniques with lower collision energies, more extensive precursor ion dissociations were observed. The detection of the corresponding fragment ions allowed the identities of guanine, PhIP or ABP, and the position of adduction to be confirmed. Some of the fragment ions of dG-C8-PhIP have not been reported by other MS/MS techniques.

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.

The objective of this research project is to develop a rapid molecular method for monitoring nitrification in a wastewater reactor. In the developed method, a monoclonal antibody (Hyb 153-3) was used because it can specifically recognize non-denatured enzymes responsible for nitrite oxidation in Nitrobacter and Nitrospira. The recognition of these enzymes under non-denatured conditions can significantly simplify the procedures of future immunoassays for environmental samples collected from various natural and engineered systems. This paper presents the ability of the selected Hyb 153-3 antibody to recognize the non-denatured form of the nitrite-oxidizing enzyme of Nitrospira in an aqueous phase as well as when the enzyme has been immobilized on a solid surface. Copyright © 2005 Society of Chemical Industry

The amplification inherent in transcription and translation of DNA has already been exploited for the development of highly sensitive immunoassays by using a reporter gene as a label that, upon in vitro expression, generates multiple enzyme molecules in solution (expression immunoassay). The most challenging task in the development of an expression immunoassay is to link the antibody to a reporter gene that also contains control elements for transcription/translation. In this work, we prepare heterobifunctional linkers that consist of a modified avidin or streptavidin covalently attached to an oligonucleotide (dA)40. (Strept)avidin interacts with a biotinylated detection antibody whereas the oligonucleotide hybridizes with a complementary poly(dT) tail added enzymically to the 3' end of the reporter gene. The linker is evaluated in a model two-site (sandwich-type) immunoassay performed in microtiter wells. A 4.3 kb plasmid containing the firefly luciferase cDNA is used as a reporter.

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.

Background Carbon nanodots (CD), a new class of carbon nanomaterials with sizes below 10 nm, have recently attracted wide attention due to their superiority in water solubility, chemical inertness, and resistance to photobleaching. As a result, CD has found important and wide applications in energy, catalysis, biological labeling, bioimaging and drug delivery. On the other hand, due to the lack of available toxicity data, there is a growing concern regarding the potential risks of CD. Hence, accurate assessment of the cytotoxicity of CD has become more important than ever before. The lactate dehydrogenase (LDH) assay is widely used to detect cytotoxicity of various nanoparticles including CD. Many recent studies used LDH assay to study the CD toxicity in various cells. However, these studies failed to further examine whether the CD were interfering with the LDH assay which would alter their findings. Findings This study investigated the possible interference of carbon nanodots on the LDH assay in human monocyte THP-1 cells. Monocytes are known to be involved in inflammable vascular diseases, and have been suggested to be the targets for CD exposure. In this study, the cytotoxicity of CD in concentrations ranging from 0.075 to 0.60 mg/mL, was determined by using the LDH assay. To validate the results of LDH assay, the cell counting method with trypan blue staining was used. With 24 hours incubation time, the cell viability of THP-1 was significantly decreased according to the trypan blue staining method. Whereas, in the LDH assay, the CD was found to interfere in a dose-dependent manner with the NADH absorbance measurements at 340 nm. Conclusions This study represents the first report on the negative interference of CD on LDH assay, and caution should be observed when evaluating the cytotoxicity of CD.

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.

Background: We report the first two-site, “sandwich type” expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase.Methods: The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3′ end of the DNA label was biotinylated and complexed with streptavidin. A sandwich immunoassay for prostate-specific antigen (PSA) was developed in which the antigen was first bound to an immobilized monoclonal antibody and then reacted with a biotinylated polyclonal antibody. The streptavidin-luciferase-coding DNA complex was then bound to the immunocomplex. The DNA label was subsequently expressed in vitro by coupled transcription and translation. The generated luciferase was measured by its characteristic bioluminescent reaction.Results: The bioluminescence was linearly related to the concentration of PSA in the sample. As low as 30 ng/L PSA was measured (12.5-μL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 μg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 μg/L; r = 0.971; n = 44).Conclusions: The use of the newly developed DNA label in a two-site immunoassay was demonstrated for the first time. The assay was applied successfully to the measurement of serum PSA.

A novel immunoassay is reported which uses an enzyme-coding DNA fragment as label (expression immunoassay). The DNA label is determined with high sensitivity by measuring the enzymatic activity produced after expression. A DNA fragment encoding the firefly luciferase is biotinylated and complexed with streptavidin. Biotinylated, specific antibodies are used for quantitation of antigen immobilized on microtiter wells. After completion of the immunoreaction, streptavidin-DNA is bound to the immunocomplex. Subsequent expression of the solid phase-bound DNA, by an one-step (coupled) cell-free transcription/translation, produces luciferase. The enzyme catalyzes the luminescent reaction of luciferin with O2 and ATP. As few as 3000 molecules of DNA label can be detected. Also, 50,000 antigen molecules can be detected, and the luminescence is a linear function of the number of antigen molecules in a range extending over 3 orders of magnitude. The high sensitivity achieved is a result of the combined amplification due to transcription/translation and the substrate turnover.

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.

Sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) of DNA is critical for obtaining high quality mass spectra. Sample impurity, solvent content, substrate surface and environmental conditions (temperature and humidity) all affect the rate of matrix–analyte co-crystallization. As a result, laser fluence threshold for desorption/ionization varies from spot to spot. When using 3-hydroxypicolinic acid (3-HPA) as the matrix, laser fluence higher than the threshold value reduces mass resolution in time-of-flight (TOF) MS as the excess energy transferred to DNA causes metastable decay. This can be overcome by either searching for ‘hot’ spots or adjusting the laser fluence. However, both solutions may require a significant amount of operator manipulation and are not ideal for automatic measurements. We have added various sugars for crystallization with the matrix to minimize the transfer of excess laser energy to DNA molecules. Fructose and fucose were found to be the most effective matrix additives. Using these additives, mass resolution for DNA molecules does not show noticeable deterioration as laser energy increases. Improved sample preparation is important for the detection of single nucleotide polymorphisms (SNPs) using primer extension with a single nucleotide. During automatic data acquisition it is difficult to routinely detect heterozygous A/T mutations, which requires resolving a mass difference of 9 Da, unless a sugar is added during crystallization.

In this report, we describe a simple and accurate method to analyze restriction fragments using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The two complementary strands of restriction fragments are separated through hybridization to a capture probe, which is a single-stranded undigested fragment. Using the biotin–streptavidin linkage, the hybrid is immobilized on streptavidin-coated magnetic beads. After conditioning the captured restriction fragments, they are eluted from the probe and their molecular weights are determined. The proposed method greatly improves the quality, and reduces the complexity of the mass spectrum by analyzing only one of the complementary strands of restriction fragments.

We report the use of a new label, an expressible enzyme-coding DNA fragment, for nucleic acid hybridization assays. The DNA label contains a firefly luciferase coding sequence downstream from a T7 RNA polymerase promoter. The target DNA (200 bp) is denatured and hybridized simultaneously with two oligonucleotide probes. One of the probes is immobilized in microtiter wells, via the digoxigenin/anti-digoxigenin interaction, and the other probe is biotinylated. After completion of the hybridization, the hybrids are reacted with a streptavidin-luciferase DNA complex. Subsequently, the solid-phase bound DNA is expressed by coupled transcription/ translation. The synthesized luciferase catalyzes the luminescent reaction of luciferin with O2 and ATP. The luminescence is linearly related to the amount of target DNA in the range of 5-5000 amol. The CVs obtained for 20 and 100 amol of target are 6.5% and 10.8%, respectively (n = 4).

MicroRNAs (miRNAs) play a crucial role in the post-transcriptional regulation of gene expression, and have already been associated with 160+ diseases. With the small sizes of miRNAs (19–25 nt) and the increasing number of human miRNAs (>2000) being reported, the accurate detection of a specific miRNA has continued to be a challenging issue. In this report, a new concept for improving the accuracy of direct mass spectrometric (MS) measurements of miRNAs is introduced. The concept aims at creating a unique signature in the mass spectrum of miRNA. To achieve this goal, one way is to capture a specific miRNA by using a complementary DNA probe. The miRNA is then eluted and digested with a specific endonuclease. The digested miRNA fragments are directly measured by using MS. The multiple peaks that correspond to the digested miRNA fragments are collectively defined as a mass signature of the miRNA being analyzed. Since the peak pattern is dependent on the intrinsic property, i.e. RNA sequence, of a specific miRNA, it is referred to as an intrinsic mass signature. Alternatively, a unique mass signature can be created by incorporating one or more extra nucleotides into the 3′ end of miRNA and the extended miRNA is measured by using MS. The molecular mass of the extended miRNA, which is defined as an extended mass signature, is expected to be different from the other miRNAs within the same sample. In comparison to matching the measured molecular mass of an undigested or unextended miRNA molecule to the expected molecular mass, the two different approaches to create a unique mass signature can improve the accuracy on qualitative MS detection of a specific miRNA.