Proteomics-Based Identification of Retinal Protein Networks Impacted by Elevated Intraocular Pressure in the Hypertonic Saline Injection Model of Experimental Glaucoma.

Elevated intraocular pressure is considered a major cause of glaucomatous retinal neurodegeneration. To facilitate a better understanding of the underlying molecular processes and mechanisms, we report a study focusing on alterations of the retina proteome by induced ocular hypertension in a rat model of the disease. Glaucomatous processes were modeled through sclerosing the aqueous outflow routes of the eyes by hypertonic saline injections into an episcleral vein. Mass spectrometry-based quantitative retina proteomics using a label-free shotgun methodology identified over 200 proteins significantly affected by ocular hypertension. Various facets of glaucomatous pathophysiology were revealed through the organization of the findings into protein interaction networks and by pathway analyses. Concentrating on retinal neurodegeneration as a characteristic process of the disease, elevated intraocular pressure-induced alterations in the expression of selected proteins were verified by targeted proteomics based on nanoflow liquid chromatography coupled with nano-electrospray ionization tandem mass spectrometry using the parallel reaction monitoring method of data acquisition. Acquired raw data are shared through deposition to the ProteomeXchange Consortium (PXD042729), making a retina proteomics dataset on the selected animal model of glaucoma available for the first time.

Synthesis and evaluation of a new class of MIF-inhibitors in activated macrophage cells and in experimental septic shock in mice.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with enzymatic activities. Anti-inflammatory effects of MIF enzyme inhibitors indicate a link between its cytokine- and catalytic activities. Herein the synthesis, docking, and bioactivity of substituted benzylidene-1-indanone and -1-tetralone derivatives as MIF-tautomerase inhibitors is reported. Many of these substituted benzylidene-1-tetralones and -indan-1-ones were potent MIF-tautomerase inhibitors (IC50 < 10 µmol/L), and the most potent inhibitors were the 1-indanone derivatives 16 and 20. Some of these compounds acted as selective enolase or ketonase inhibitors. In addition, compounds 16, 20, 26, 37 and 61 efficiently inhibited NO, TNFα and IL-6 production in lipopolysaccharide-induced macrophages. Compound 20, 37 and 61 also inhibited ROS generation, and compound 26 and 37 abolished activation of NF-κB. Compound 37 significantly augmented hypothermia induced by high dose of lipopolysaccharide in mice. The possible mechanisms of action were explored using molecular modelling and docking, as well as molecular dynamics simulations.

Mass spectrometry-based retina proteomics.

A subfield of neuroproteomics, retina proteomics has experienced a transformative growth since its inception due to methodological advances in enabling chemical, biochemical, and molecular biology techniques. This review focuses on mass spectrometry's contributions to facilitate mammalian and avian retina proteomics to catalog and quantify retinal protein expressions, determine their posttranslational modifications, as well as its applications to study the proteome of the retina in the context of biology, health and diseases, and therapy developments.

Global Comparative Label-Free Yeast Proteome Analysis by LC-MS/MS After High-pH Reversed-Phase Peptide Fractionation Using Solid-Phase Extraction Cartridges.

Discovery-driven comparative proteomics employing the bottom-up strategy with label-free quantification on high-resolution mass analyzers like an Orbitrap in a hybrid instrument has the capacity to reveal unique biological processes in the context of plant metabolic engineering. However, proteins are very heterogeneous in nature with a wide range of expression levels, and overall coverage may be suboptimal regarding both the number of protein identifications and sequence coverage of the identified proteins using conventional data-dependent acquisitions without sample fractionation before online nanoflow liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS). In this chapter, we detail a simple and robust method employing high-pH reversed-phase (HRP) peptide fractionation using solid-phase extraction cartridges for label-free proteomic analyses. Albeit HRP fractionation separates peptides according to their hydrophobicity like the subsequent nanoflow gradient reversed-phased LC relying on low pH mobile phase, the two methods are orthogonal. Presented here as a protocol with yeast (Saccharomyces cerevisiae) as a frequently used model organism and hydrogen peroxide to exert cellular stress and survey its impact compared to unstressed control as an example, the described workflow can be adapted to a wide range of proteome samples for applications to plant metabolic engineering research.

Proteomics-Based Retinal Target Engagement Analysis and Retina-Targeted Delivery of 17ß-Estradiol by the DHED Prodrug for Ocular Neurotherapy in Males.

We examined the impact of 17ß-estradiol (E2) eye drops on the modulation of the proteome profile in the male rat retina. With discovery-driven proteomics, we have identified proteins that were regulated by our treatment. These proteins were assembled to several bioinformatics-based networks implicating E2's beneficial effects on the male rat retina in a broad context of ocular neuroprotection including the maintenance of retinal homeostasis, facilitation of efficient disposal of damaged proteins, and mitochondrial respiratory chain biogenesis. We have also shown for the first time that the hormone's beneficial effects on the male retina can be constrained to this target site by treatment with the bioprecursor prodrug, DHED. A large concentration of E2 was produced after DHED eye drops not only in male rat retinae but also in those of rabbits. However, DHED treatment did not increase circulating E2 levels, thereby ensuring therapeutic safety in males. Targeted proteomics focusing on selected biomarkers of E2's target engagement further confirmed the prodrug's metabolism to E2 in the male retina and indicated that the retinal impact of DHED treatment was identical to that of the direct E2 treatment. Altogether, our study shows the potential of topical DHED therapy for an efficacious and safe protection of the male retina without the unwanted hormonal side-effects associated with current estrogen therapies.

Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications.

The widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17ß-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.

Comparative Proteomics Analysis Reveals Unique Early Signaling Response of Saccharomyces cerevisiae to Oxidants with Different Mechanism of Action.

The early signaling events involved in oxidant recognition and triggering of oxidant-specific defense mechanisms to counteract oxidative stress still remain largely elusive. Our discovery driven comparative proteomics analysis revealed unique early signaling response of the yeast Saccharomyces cerevisiae on the proteome level to oxidants with a different mechanism of action as early as 3 min after treatment with four oxidants, namely H2O2, cumene hydroperoxide (CHP), and menadione and diamide, when protein abundances were compared using label-free quantification relying on a high-resolution mass analyzer (Orbitrap). We identified significant regulation of 196 proteins in response to H2O2, 569 proteins in response to CHP, 369 proteins in response to menadione and 207 proteins in response to diamide. Only 17 proteins were common across all treatments, but several more proteins were shared between two or three oxidants. Pathway analyses revealed that each oxidant triggered a unique signaling mechanism associated with cell survival and repair. Signaling pathways mostly regulated by oxidants were Ran, TOR, Rho, and eIF2. Furthermore, each oxidant regulated these pathways in a unique way indicating specificity of response to oxidants having different modes of action. We hypothesize that interplay of these signaling pathways may be important in recognizing different oxidants to trigger different downstream MAPK signaling cascades and to induce specific responses.

Retina-Targeted Delivery of 17ß-Estradiol by the Topically Applied DHED Prodrug.

The purpose of this study was to explore retina-targeted delivery of 17ß-estradiol (E2), a powerful neuroprotectant, by its bioprecursor prodrug 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED) administered as eye drops in animal models. Compared to the parent hormone, DHED displayed increased transcorneal flux ex vivo both with and without the presence of 2-hydroxypropyl-ß-cyclodextrin used as a penetration-enhancing excipient in rat, rabbit, and pig. In vitro, the prodrug also showed facile bioactivation to E2 in the retina but not in the cornea. After topical administration to rats and rabbits, peak DHED-derived E2 concentrations reached 13 ± 5 ng/g and 18 ± 7 ng/g in the retina of female rats and rabbits, respectively. However, the prodrug remained inert in the rest of the body and, therefore, did not cause increase in circulating hormone concentration, as well as wet uterine and anterior pituitary weights as typical markers of E2's endocrine impact. Altogether, our studies presented here have demonstrated the premise of topical retina-selective estrogen therapy by the DHED prodrug approach for the first time and provide compelling support for further investigation into the full potential of DHED for an efficacious and safe ocular neurotherapy.

17ß-Estradiol Delivered in Eye Drops: Evidence of Impact on Protein Networks and Associated Biological Processes in the Rat Retina through Quantitative Proteomics.

To facilitate the development of broad-spectrum retina neuroprotectants that can be delivered through topical dosage forms, this proteomics study focused on analyzing target engagements through the identification of functional protein networks impacted after delivery of 17ß-estradiol in eye drops. Specifically, the retinae of ovariectomized Brown Norway rats treated with daily eye drops of 17ß-estradiol for three weeks were compared to those of vehicle-treated ovariectomized control animals. We searched the acquired raw data against a composite protein sequence database by using Mascot, as well as employed label-free quantification to detect changes in protein abundances. Our investigation using rigorous validation criteria revealed 331 estrogen-regulated proteins in the rat retina (158 were up-regulated, while 173 were down-regulated by 17ß-estradiol delivered in eye drops). Comprehensive pathway analyses indicate that these proteins are relevant overall to nervous system development and function, tissue development, organ development, as well as visual system development and function. We also present 18 protein networks with associated canonical pathways showing the effects of treatments for the detailed analyses of target engagements regarding potential application of estrogens as topically delivered broad-spectrum retina neuroprotectants. Profound impact on crystallins is discussed as one of the plausible neuroprotective mechanisms.

Development and application of sub-2-µm particle CO2 -based chromatography coupled to mass spectrometry for comprehensive analysis of lipids in cottonseed extracts.

RATIONALE: Refined cottonseed oil has widespread applications in the food and chemical industries. Although the major lipids comprising cottonseed oil (triacylglycerols) are well known, there are many diverse lipid species in cotton seeds that occur at much lower levels and have important nutritional or anti-nutritional properties. METHODS: The lipid technical samples were prepared in chloroform. The biological samples were extracted using a mixture of isopropanol/chloroform/H2 O (2:1:0.45). The data were collected using high and low collision energy with simultaneous data collection on a time-of-flight (TOF) mass spectrometer which allowed the characterization of lipids by precursor and product ion alignment. The supercritical fluid chromatography methodology is flexible and can be altered to provide greater retention and separation. The comprehensive method was used to screen seed lipid extracts from several cotton genotypes using multivariate statistical analysis. RESULTS: Method variables influencing the peak integrity and chromatographic separation for a mixture of lipids with different degrees of polarity were explored. The experiments were designed to understand the chromatographic behavior of lipids in a controlled setting using a variety of lipid extracts. Influences of acyl chain length and numbers of double bonds were investigated using single moiety standards. CONCLUSIONS: The methodology parameters were examined using single moiety lipid standards and standard mixtures. The method conditions were applied to biological lipid extracts, and adjustments were investigated to manipulate the chromatography. Insights from these method variable manipulations will help to frame the development of targeted lipid profiling and screening protocols. Copyright © 2017 John Wiley & Sons, Ltd.

Glucose induces sensitivity to oxygen deprivation and modulates insulin/IGF-1 signaling and lipid biosynthesis in Caenorhabditis elegans.

Diet is a central environmental factor that contributes to the phenotype and physiology of individuals. At the root of many human health issues is the excess of calorie intake relative to calorie expenditure. For example, the increasing amount of dietary sugars in the human diet is contributing to the rise of obesity and type 2 diabetes. Individuals with obesity and type 2 diabetes have compromised oxygen delivery, and thus it is of interest to investigate the impact a high-sugar diet has on oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, we determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide synthesis, modulates anoxia survival. Additionally, a glucose-supplemented diet alters lipid localization and initiates a positive chemotaxis response. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified show homology to human genes that are differentially regulated in response to obesity or type 2 diabetes, suggesting that there may be conserved gene expression responses between C. elegans fed a glucose-supplemented diet and a diabetic and/or obesity state observed in humans. These findings support the utility of the C. elegans model for understanding the molecular mechanisms regulating dietary-induced metabolic diseases.

Characterization of Mycobacterium africanum subtype I among cows in a dairy farm in Bangladesh using spoligotyping.

Acid-fast bacilli (AFB) were detected in the autopsy lung tissue homogenate samples of four cows (variety Frisian cross) in a dairy farm in Bangladesh. Histopathological examination of the lung tissue demonstrated prominent granulomas, caseating necrosis and calcification indicative of tuberculosis (TB) infection. Mycobacteria could not be cultured from the tissue homogenate samples by Lowenstein-Jensen based conventional culture method though AFB were evident by Ziehl-Neelsen (ZN) staining of the smears of tissue homogenate and in paraffin embedded tissue slices. Spoligotyping performed on DNA extracts of paraffin embedded lung tissue samples confirmed the AFB as a member of the M. tuberculosis complex (MTBC) with a pattern assigned to M. africanum subtype I. This characterization by spoligotyping was confirmed by subjecting M. africanum subtype I isolates from other parts of the world to an alternative identification method based on DNA polymorphism in the gyrB gene (Hain Life Science, GmbH, Nehren, Germany). Since M. africanum is believed to be a human pathogen, general infection in cattle may be a public health threat. The presence of these bacteria in the animal reservoir most likely originated from a caretaker.

Assessment of population structure and major circulating phylogeographical clades of Mycobacterium tuberculosis complex in Bangladesh suggests a high prevalence of a specific subclade of ancient M. tuberculosis genotypes.

Spoligotyping was performed to study the population structure of Mycobacterium tuberculosis complex strains (n = 224) from Bangladesh. Strains were split into principal genetic group 1 (PGG 1 [75.0%]) and PGG 2 and 3 (25%). Forty-nine strains with a new spoligotype signature and considered as south or southeast Asian-linked emerging clones were designated as "Matlab type."