Moxifloxacin Concentration and Proteomic Analysis of Aqueous Humor in Human Uveitis Associated with Oral Moxifloxacin Therapy.

PURPOSE: The aim was to report the aqueous humor moxifloxacin concentration and proteome profile of an individual with bilateral uveitis-like syndrome with pigment dispersion. METHODS: Multiple reactions monitoring mass spectrometry quantified the aqueous concentration of moxifloxacin in the affected individual. Shotgun proteomic analysis performed via liquid chromatography tandem mass spectrometry (LC-MS/MS) defined the protein profile in the affected individual and unaffected control samples. RESULTS: Moxifloxacin was present at higher than expected levels in aqueous humor 18 days following oral administration. One-third of the proteins were identified by significantly lower spectral counts in the aqueous of the individual with moxifloxacin associated uveitis compared to the unaffected control. CONCLUSION: Moxifloxacin was detected in aqueous humor 18 days following the completion of oral administration. These results suggest that moxifloxacin toxicity may be responsible for the uveitis-like syndrome with pigment dispersion syndrome induced by moxifloxacin therapy.

Exploring the Bone Proteome to Help Explain Altered Bone Remodeling and Preservation of Bone Architecture and Strength in Hibernating Marmots.

Periods of physical inactivity increase bone resorption and cause bone loss and increased fracture risk. However, hibernating bears, marmots, and woodchucks maintain bone structure and strength, despite being physically inactive for prolonged periods annually. We tested the hypothesis that bone turnover rates would decrease and bone structural and mechanical properties would be preserved in hibernating marmots (Marmota flaviventris). Femurs and tibias were collected from marmots during hibernation and in the summer following hibernation. Bone remodeling was significantly altered in cortical and trabecular bone during hibernation with suppressed formation and no change in resorption, unlike the increased bone resorption that occurs during disuse in humans and other animals. Trabecular bone architecture and cortical bone geometrical and mechanical properties were not different between hibernating and active marmots, but bone marrow adiposity was significantly greater in hibernators. Of the 506 proteins identified in marmot bone, 40 were significantly different in abundance between active and hibernating marmots. Monoaglycerol lipase, which plays an important role in fatty acid metabolism and the endocannabinoid system, was 98-fold higher in hibernating marmots compared with summer marmots and may play a role in regulating the changes in bone and fat metabolism that occur during hibernation.

Effects of cyclin-dependent kinase inhibitor Purvalanol B application on protein expression and developmental progression in intra-erythrocytic Plasmodium falciparum parasites.

BACKGROUND: The 2013 Malaria World Report indicated that in 2012 there were approximately 207 million cases of malaria, which resulted in an estimated 627,000 malaria-related deaths. Due to the alarming resistance of these parasites to traditional anti-malarial treatments there is a pressing need to not only identify new anti-malarial compounds, but also to characterize the effect of compounds known to have an effect on the parasite life cycle. This study reports on effects of kinase inhibitor Purvalanol B administration on the growth and protein expression of Plasmodium falciparum late-stage trophozoites. METHODS: A SYBR® Green I parasite growth assay was used to measure the IC50 of Purvalanol B with P. falciparum (strain W2). Purvalanol B or DMSO control were applied to synchronized parasites 36 hours post invasion and parasites were incubated for 12 hours. Giemsa-stained blood smears were used to determine the effect of Purvalanol B on parasite growth, global quantitative proteomic analysis was used to examine differences in protein expression between Purvalanol B-treated and control parasites and results were confirmed by qPCR. RESULTS: There were no differences in parasitaemia between inhibitor-treated and control parasites. However, the ability of Purvalanol B-treated parasites to form schizonts was significantly reduced. Proteomic analysis detected 76 human proteins and 518 P. falciparum proteins (63 in control cultures only, 56 proteins in Purvalanol B cultures only, and 399 proteins in both cultures). Quantitative analysis of protein extracts revealed eight proteins that were up-regulated in the inhibitor-treated cultures, including several components of the parasite's proteasome complex and thioredoxin reductase. Two proteins appeared to be down-regulated, including a helicase and an RNA-binding protein. CONCLUSION: Purvalanol B application decreases the ability of late-stage P. falciparum trophozoites to form multinucleated schizonts and up-regulates proteasome subunits and proteins that contribute to redox homeostasis, which may indicate an increase in oxidative stress as a result of inhibitor application. While the efficacy of Purvalanol B is relatively low for use as an anti-malarial therapy, quantitative proteomic analysis may serve as a method of examining the action of drugs on the parasite and indicate the likelihood of future resistance development.

Proteomic Profiling of Sugar Beet (Beta vulgaris) Leaves during Rhizomania Compatible Interactions.

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), severely impacts sugar beet (Beta vulgaris) production throughout the world, and is widely prevalent in most production regions. Initial efforts to characterize proteome changes focused primarily on identifying putative host factors that elicit resistant interactions with BNYVV, but as resistance breaking strains become more prevalent, effective disease control strategies will require the application of novel methods based on better understanding of disease susceptibility and symptom development. Herein, proteomic profiling was conducted on susceptible sugar beet, infected with two strains of BNYVV, to clarify the types of proteins prevalent during compatible virus-host plant interactions. Total protein was extracted from sugar beet leaf tissue infected with BNYVV, quantified, and analyzed by mass spectrometry. A total of 203 proteins were confidently identified, with a predominance of proteins associated with photosynthesis and energy, metabolism, and response to stimulus. Many proteins identified in this study are typically associated with systemic acquired resistance and general plant defense responses. These results expand on relatively limited proteomic data available for sugar beet and provide the ground work for additional studies focused on understanding the interaction of BNYVV with sugar beet.

Mass spectrometry contamination from Tinuvin 770, a common additive in laboratory plastics.

The superior sensitivity of current mass spectrometers makes them prone to contamination issues, which can have deleterious effects on sample analysis. Here, bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate (marketed under the name Tinuvin 770) is identified as a major contaminant in applications using liquid chromatography coupled with mass spectrometry (LC-MS). Tinuvin 770 is often added to laboratory and medical plastics as a UV stabilizer. One particular lot of microcentrifuge tubes was found to have an excess of this compound that would leach into samples and drastically interfere with LC-MS data acquisition. Further analysis found that Tinuvin 770 readily leached into polar and nonpolar solvents from the contaminated tube lot. Efforts to remove Tinuvin 770 from contaminated samples were unsuccessful. A prescreening method using MALDI-TOF MS is presented to prevent system contamination and sample loss.

Multiplexed analysis of steroid hormones in human serum using novel microflow tile technology and LC-MS/MS.

A novel microfluidic chromatography device coupled with tandem mass spectrometry (LC-MS/MS) was utilized for the multiplex analysis of 5 steroids (testosterone, dihydrotestosterone, progesterone, cortisol, cortisone) in human serum. The use of microfluidics allowed for reduction of the chromatographic flow rate to 3µl/min with overall method run times comparable to standard flow LC-MS/MS methods reported in the literature, corresponding to a 150 fold decrease in solvent consumption. Furthermore, a simple sample preparation protocol was employed requiring injection of only 0.5µl of sample, corresponding to a 100-400 fold increase in on-column sensitivity as compared to published standard flow assays. The measured LOQ for both testosterone and progesterone was 0.4ng/mL, representing an improvement over reported literature values obtained by standard flow methods employing comparable sample preparation and large injection volumes. The LOQs for cortisol (1.9ng/mL), cortisone (0.3ng/mL), and dihydrotestosterone (1.4ng/mL) were all within a biologically relevant range. A comparison of clinical serum samples was performed for the analysis of testosterone using this microfluidic LC-MS/MS assay and the Beckman Access II automated antibody-based measurement system. The immunoassay results were systematically higher due to matrix interference which was easily resolved with the increased chromatographic resolution obtained in the microflow LC-MS/MS assay.

Comparative proteomic profiling of patients with atopic dermatitis based on history of eczema herpeticum infection and Staphylococcus aureus colonization.

BACKGROUND: Atopic dermatitis (AD) is the most common inflammatory skin disorder in the general population worldwide, and the majority of patients are colonized with Staphylococcus aureus. Eczema herpeticum is a disseminated herpes simplex virus infection that occurs in a small subset of patients. OBJECTIVES: The goal was to conduct proteomic profiling of patients with AD based on S. aureus colonization status and history of eczema herpeticum. We hoped to identify new biomarkers for improved diagnosis and prediction of eczema herpeticum and S. aureus susceptibility and to generate new hypotheses regarding disease pathogenesis. METHODS: Skin taping was performed on nonlesional skin of nonatopic control subjects and on lesional and nonlesional skin of patients with AD. Subjects were classified according to the history of eczema herpeticum and S. aureus colonization. Proteins were analyzed by using mass spectrometry; diagnostic groups were compared for statistically significant differences in protein expression. RESULTS: Proteins related to the skin barrier (filaggrin-2, corneodesmosin, desmoglein-1, desmocollin-1, and transglutaminase-3) and generation of natural moisturizing factor (arginase-1, caspase-14, and gamma-glutamyl cyclotransferase) were expressed at significantly lower levels in lesional versus nonlesional sites of patients with AD with and without history of eczema herpeticum; epidermal fatty acid-binding protein was expressed at significantly higher levels in patients with methicillin-resistant S. aureus. CONCLUSION: This noninvasive, semiquantitative profiling method has revealed novel proteins likely involved in the pathogenesis of AD. The lower expression of skin barrier proteins and enzymes involved in the generation of the natural moisturizing factor could further exacerbate barrier defects and perpetuate water loss from the skin. The greater expression of epidermal fatty acid-binding protein, especially in patients colonized with methicillin-resistant S. aureus, might perpetuate the inflammatory response through eicosanoid signaling.

Proteomics methods and applications for the practicing clinician.

OBJECTIVE: To describe clinical proteomics from discovery techniques and their limitations, to applications in allergy, asthma, and immunology, and finally to how proteomics can be integrated into clinical practice. DATA SOURCES: Despite many inherent challenges, proteomics-based methods have become a powerful and popular means of profiling clinical samples for the purpose of biomarker discovery. Although several strategies exist, clinical proteomics for the purpose of biomarker discovery generally focuses on 1 of 3 basic workflows: (1) 2-dimensional gel electrophoresis to quantitate relative protein levels followed by mass spectrometry (MS) to identify proteins of interest, (2) non-gel-based methods that rely on liquid chromatography MS (LCMS) for both quantitation and identification of proteins, and (3) protein profiling methods that do not directly result in the identification of proteins but rather generate "fingerprints" that are compared among individuals or samples. STUDY SELECTION: Regardless of the strategy being pursued, a few general experimental steps are followed that will be expounded on in the text. These proteomics techniques have been applied to discover new biomarkers in biofluids and tissues from individuals with a variety of conditions, including allergy, asthma, atopic dermatitis, inflammatory diseases, chronic obstructive pulmonary disease, and other lung diseases. RESULTS: After biomarker discovery, LCMS-based proteomics offers several advantages over traditional antibody-based clinical assays, including greater specificity, cost- and time-effectiveness, and the potential to multiplex up to hundreds of peptides in a single assay. CONCLUSION: With many guidelines now in place and model studies on which to design future experiments, there is reason to be optimistic that candidate protein biomarkers will be discovered using proteomics and translated into clinical assays.

Probing the control elements of the CYP1A1 switching module in H4IIE hepatoma cells.

Previous research from our laboratory has shown a switch-like response to PCB 126 mediated CYP1A1 induction in primary rat hepatocytes and in H4IIE rat hepatoma cells. On a single cell level, cells appear to be either "on" or "off" for CYP1A1 induction at a given dose; some cells never respond to PCB 126. These cells represent a non-responding population. Cells that are switched "on" by PCB 126 display varying levels of induction, much like the dimmer on a light switch. The goal of the present research is to begin to uncover the mechanism for this switch-like response to CYP1A1 induction in H4IIE rat hepatoma cells. The AhR pathway is modulated by multiple co-activators and by phosphorylation. This research focuses on the phosphorylation cascades initiated by PCB 126 and the role they play in CYP1A1 induction. Our research reveals a likely role for protein kinase C (PKC) in this switch response. Inhibition of PKC by H-7 dramatically reduced the percent of cells that express CYP1A1 in response to PCB 126 treatment, as determined by flow cytometry. The effect of H-7 was concentration dependent, decreasing the number of cells expressing CYP1A1 rather than decreasing the level of CYP1A1 in all cells. This finding provides further evidence for the switch-like behavior of CYP1A1 induction and implicates PKC in this response to PCB126. The protein kinase inhibitor, HA-1004, had only a minor effect on CYP1A1 induction. A high-throughput immunoblot screen for 40 proteins revealed the regulation of several proteins/phosphoproteins by PCB 126. Most importantly, two proteins containing phosphoserine/phoshothreonine residues were increased by PCB126 treatment. However, PKC translocation studies and activity studies failed to verify that PCB126 activates PKC. It is possible that constitutive PKC activity is sufficient to maintain phosphorylation of critical components of the AhR pathway. Immunoblotting studies showed that MAP kinases ERK and JNK are not activated by PCB 126 in H4IIE cells and the ERK inhibitor U0126 did not impair CYP1A1 induction. Additional studies are planned to further investigate the role of PKC in the switch-like response to PCB 126.

Regional induction of CYP1A1 in rat liver following treatment with mixtures of PCB 126 and PCB 153.

Liver enzyme induction has been shown previously to be regional with clear borders between induced and uninduced regions in vivo, and cells either fully induced or not induced in vitro. The current study examined this phenomenon in vivo by evaluating enzyme induction after exposure to PCB 126 and PCB 153 in female Fisher 344 (F344) and male Sprague-Dawley (SD) rats. IHC revealed a regional induction of CYP1A1 after exposure to PCB 126, apparent in the centrilobular region at lower doses and progressing to panlobular with higher doses. PCB 153 exposure induced CYP2B1/2 in the centrilobular region, which spread to the midzonal region as the dose increased, but never became panlobular even at the highest dosage tested. In rats treated with PCB 126 in combination with high doses of PCB 153, induction of CYP1A1 occurred preferentially in the periportal region, a reversal from the pattern seen with PCB 126 alone. This CYP1A1 induction pattern reversal is a unique example of complex biological interactions between coplanar (PCB 126) and noncoplanar (PCB 153) halogenated aromatic hydrocarbons.

Single cell analysis of switch-like induction of CYP1A1 in liver cell lines.

The shape of the dose-response curve may vary depending on whether one examines response at a population or a single cell level. Populations of cells may exhibit a graded response whereas single cell responses may have threshold or switch-like behavior. Studies in vivo and in vitro using primary hepatocyte cultures have shown that induction of CYP1A1 in the liver exhibits switch-like behavior in response to PCB 126 (3,3',4,4',5-pentachlorobiphenyl). The goal of the present study was to determine if two liver cell lines (H4IIE rat hepatoma and Hepa 1c1c7 mouse hepatoma) also show switch-like behavior and develop experimental models for studying mechanisms of these switch-like responses. Both cell lines were analyzed via concentration-response and time-course studies using quantitative real-time PCR, revealing a sigmoidal concentration-response curve for CYP1A1 mRNA induction at the population level. To study CYP1A1 protein induction on a single cell level, flow cytometry was employed. In both cell lines the distribution of fluorescence increased with increasing concentrations of PCB 126. The switch behavior was more pronounced in the H4IIE cells than in the Hepa 1c1c7 cells, exhibiting a well-defined shift of induction from the "off" to the "on" state. The concentration-response curve at the single cell level appeared more switch-like with two populations of cells-basal levels and maximally induced. Immunocytochemistry studies of individual cells also support these conclusions. Our data support the hypothesis that PCB 126 induces CYP1A1 in a switch-like fashion in H4IIE rat hepatoma cells. These cells can now be used to study the mechanism of the biological switch.