Characterization and Encapsulation of Natural Antioxidants: Interaction, Protection and Delivery

This book focuses on the prediction of antioxidant peptides and the improvement of the aqueous solubility, chemical stability, bioactivity, and release properties of natural antioxidants through enzymatic glycosylation and encapsulation technology. The rational design and construction of delivery systems are effective for the application of antioxidants in the industry. Special attention in this book is given to inorganic nanoparticles, biopolymer-based particulate systems, prediction in antioxidant activity of peptides, evaluation of encapsulation and delivery performance, and application in commercial products

Incorporating standardised drift-tube ion mobility to enhance non-targeted assessment of the wine metabolome (LC×IM-MS)

Liquid chromatography with drift-tube ion mobility spectrometry-mass spectrometry (LCxIM-MS) is emerging as a powerful addition to existing LC-MS workflows for addressing a diverse range of metabolomics-related questions [1,2]. Importantly, excellent precision under repeatability and reproducibility conditions of drift-tube IM separations [3] supports the development of non-targeted approaches for complex metabolome assessment such as wine characterisation [4]. In this work, fundamentals of this new analytical metabolomics approach are introduced and application to the analysis of 90 authentic red and white wine samples originating from Macedonia is presented. Following measurements, intersample alignment of metabolites using non-targeted extraction and three-dimensional alignment of molecular features (retention time, collision cross section, and high-resolution mass spectra) provides confidence for metabolite identity confirmation. Applying a fingerprinting metabolomics workflow allows statistical assessment of the influence of geographic region, variety, and age. This approach is a state-of-the-art tool to assess wine chemodiversity and is particularly beneficial for the discovery of wine biomarkers and establishing product authenticity based on development of fingerprint libraries

Selected Papers from XVI MaNaPro and XI ECMNP

The oceans harbor the majority of the Earth´s biodiversity. Marine organisms/microorganisms provide a diverse array of natural products, which are important sources of biologically active agents with unique chemical structures and a broad range of medical and biotechnological applications. The XVI MaNaPro and XI ECMNP conferences aim to present advances and future perspectives on marine natural product research to the scientific community by gathering scientists who work in marine chemistry and related scientific fields from all over the world and at different seniority levels. This Special Issue was organized on the occasion of the 2nd joint XVI MaNaPro and XI ECMNP meeting (http://wmnp2019.ipleiria.pt/) held in Peniche, Portugal, in 2019. It comprises 12 original research articles that exemplify research performed in the scope of the conference topic

Computational Tools for the Untargeted Assignment of FT-MS Metabolomics Datasets

Metabolomics is the study of metabolomes, the sets of metabolites observed in living systems. Metabolism interconverts these metabolites to provide the molecules and energy necessary for life processes. Many disease processes, including cancer, have a significant metabolic component that manifests as differences in what metabolites are present and in what quantities they are produced and utilized. Thus, using metabolomics, differences between metabolomes in disease and non-disease states can be detected and these differences improve our understanding of disease processes at the molecular level. Despite the potential benefits of metabolomics, the comprehensive investigation of metabolomes remains difficult. A popular analytical technique for metabolomics is mass spectrometry. Advances in Fourier transform mass spectrometry (FT-MS) instrumentation have yielded simultaneous improvements in mass resolution, mass accuracy, and detection sensitivity. In the metabolomics field, these advantages permit more complicated, but more informative experimental designs such as the use of multiple isotope-labeled precursors in stable isotope-resolved metabolomics (SIRM) experiments. However, despite these potential applications, several outstanding problems hamper the use of FT-MS for metabolomics studies. First, artifacts and data quality problems in FT-MS spectra can confound downstream data analyses, confuse machine learning models, and complicate the robust detection and assignment of metabolite features. Second, the assignment of observed spectral features to metabolites remains difficult. Existing targeted approaches for assignment often employ databases of known metabolites; however, metabolite databases are incomplete, thus limiting or biasing assignment results. Additionally, FT-MS provides limited structural information for observed metabolites, which complicates the determination of metabolite class (e.g. lipid, sugar, etc. ) for observed metabolite spectral features, a necessary step for many metabolomics experiments. To address these problems, a set of tools were developed. The first tool identifies artifacts with high peak density observed in many FT-MS spectra and removes them safely. Using this tool, two previously unreported types of high peak density artifact were identified in FT-MS spectra: fuzzy sites and partial ringing. Fuzzy sites were particularly problematic as they confused and reduced the accuracy of machine learning models trained on datasets containing these artifacts. Second, a tool called SMIRFE was developed to assign isotope-resolved molecular formulas to observed spectral features in an untargeted manner without a database of expected metabolites. This new untargeted method was validated on a gold-standard dataset containing both unlabeled and 15N-labeled compounds and was able to identify 18 of 18 expected spectral features. Third, a collection of machine learning models was constructed to predict if a molecular formula corresponds to one or more lipid categories. These models accurately predict the correct one of eight lipid categories on our training dataset of known lipid and non-lipid molecular formulas with precisions and accuracies over 90% for most categories. These models were used to predict lipid categories for untargeted SMIRFE-derived assignments in a non-small cell lung cancer dataset. Subsequent differential abundance analysis revealed a sub-population of non-small cell lung cancer samples with a significantly increased abundance in sterol lipids. This finding implies a possible therapeutic role of statins in the treatment and/or prevention of non-small cell lung cancer. Collectively these tools represent a pipeline for FT-MS metabolomics datasets that is compatible with isotope labeling experiments. With these tools, more robust and untargeted metabolic analyses of disease will be possible

Morphological, cellular and proteomic features of canine myxomatous mitral valve disease

Myxomatous mitral valve degeneration (MMVD) is the single most common cardiac disease of the dog, and is analogous to Mitral Valve Prolapse in humans. Very little is known about the aetiopathogenesis of this disease or the changes in valvular interstitial cell populations in diseased valves. The aim of this study was to identify morphological, cellular and molecular changes associated with MMVD. Mitral valve leaflets from both normal and varying grades (Whitney’s 1-4) of diseased dogs were subject to image analysis, immunophenotyping, proteomics and RT-PCR. Image analysis - leaflet thickening due to accumulation of glycosaminoglycan was significant in this disease. MMVD is associated with loss of connective tissue, reduction in cell numbers but no change in cell shape in the overtly myxomatous area. Near the surface, increase in valvular interstitial cells (VIC) towards the damaged endothelium in concert with destruction of collagen and building up of ground substance was manifested during the disease process. Immunophenotyping - activated myofibroblasts were increased and fibroblast-like VICs were reduced without any change in desmin and myosin expression in MMVD compared to clinical normal dogs. In addition, other cell types like macrophage, adipocyte, chondrocyte, mast cell, and stem cell were identified and their possible role in MMVD is discussed. Proteomics - a protein expression profile was established, with 64 proteins being positively identified from dog’s mitral valve using 1-D SDS PAGE LC/MS. Amongst them 44 proteins were differentially expressed comparing normal and severely diseased. Two actin binding proteins, tropomyosin alpha and myosin light chain-2 were found to be differentially expressed in the normal but down regulated in the diseased. RT-PCR was used to assess the expression of 8 genes of interest. Their expression was compared with 3 different housekeeping genes

Role of adipose tissue in the pathogenesis and treatment of metabolic syndrome

© Springer International Publishing Switzerland 2014. Adipocytes are highly specialized cells that play a major role in energy homeostasis in vertebrate organisms. Excess adipocyte size or number is a hallmark of obesity, which is currently a global epidemic. Obesity is not only the primary disease of fat cells, but also a major risk factor for the development of Type 2 diabetes, cardiovascular disease, hypertension, and metabolic syndrome (MetS). Today, adipocytes and adipose tissue are no longer considered passive participants in metabolic pathways. In addition to storing lipid, adipocytes are highly insulin sensitive cells that have important endocrine functions. Altering any one of these functions of fat cells can result in a metabolic disease state and dysregulation of adipose tissue can profoundly contribute to MetS. For example, adiponectin is a fat specific hormone that has cardio-protective and anti-diabetic properties. Inhibition of adiponectin expression and secretion are associated with several risk factors for MetS. For this purpose, and several other reasons documented in this chapter, we propose that adipose tissue should be considered as a viable target for a variety of treatment approaches to combat MetS

UVR8 mediated spatial differences as a prerequisite for UV-B induced inflorescence phototropism

In Arabidopsis hypocotyls, phototropins are the dominant photoreceptors for the positive phototropism response towards unilateral ultraviolet-B (UV-B) radiation. We report a stark contrast of response mechanism with inflorescence stems with a central role for UV RESISTANCE LOCUS 8 (UVR8). The perception of UV-B occurs mainly in the epidermis and cortex with a lesser contribution of the endodermis. Unilateral UV-B exposure does not lead to a spatial difference in UVR8 protein levels but does cause differential UVR8 signal throughout the stem with at the irradiated side 1) increase of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), 2) an associated strong activation of flavonoid biosynthesis genes and flavonoid accumulation, 3) increased GA2oxidase expression, diminished gibberellin1 levels and accumulation of DELLA protein REPRESSOR OF GA1 (RGA) and, 4) increased expression of the auxin transport regulator, PINOID, contributing to local diminished auxin signalling. Our molecular findings are in support of the Blaauw theory (1919), suggesting that differential growth occurs trough unilateral photomorphogenic growth inhibition. Together the data indicate phototropin independent inflorescence phototropism through multiple locally UVR8-regulated hormone pathways

Regulatory role of ambient pH in the expression of pathogenicity determinant gene products of Beauveria bassiana and Metarhizium anisopliae

Entomopathogenic fungi (EPF) are the one of the potential cause of the morbidity and mortality of insects. In agro-forestry uses, they are applied mainly in the form of conidial preparations in dry, aqueous or oil formulations. This approach, while practical, works in a hit and miss fashion leading to a frustrating dilemma of why successes and failure perpetuate. The fundamental solution is to bridge gaps in our knowledge about conidia of EPF in varied environments where they confront a diversity of insect hosts to start their pathogenesis.This thesis was undertaken to examine the effects of hydration and the regulatory role of ambient pH on proteases which are the primary pathogenicity determinants in Beauveria bassiana and Metarhizium anisopliae. The approaches used were those of biochemical, proteomics and functional proteomics. Novel aspects of pH regulation/homeostasis during the soaking of conidia in water, (type II water, which had a maximum electrical conductivity of 1ìS/ cm at 298K/ 25° C) were identified. Hydrated conidia showed swelling in type II water as assessed by (Multisizer IIITM (Coulter CounterTM). Release of proteases, metabolic activity through liberation of ammonia and citrate and synthesis of protein, RNA and DNA was established. It was deduced that conidial enzymes are either attached by loose hydrogen bonding or were associated to the spore membranes. Water soaked or hydrated conidia can secrete citrate and ammonia to modify the ambient pH and maximize the activity of secreted proteases. Pr1- and- Pr2-like proteases were liberated by washing conidia in tween (Tw), water (Ww) and buffer. The washing of conidia in buffers (pH 4-10) affected the release/activity of Pr1 and Pr2. The thesis shows a newly designed native IPG strip zymography to identify the release of 4 and 8 isoforms of proteases, respectively from conidia. The 2-DE zymography (copolymerized gelatin) of protease from Tw of B. bassiana and M. anisopliae indicated one band (Mr 70 kDa; pI 6.3) and six isozymes (Mr 115-129 kDa; pI 3.7-9.0), respectively, which were identified using mass spectrometry (MALDI-TOF) as a serine-like protease. Six metalloprotease isozymes from M. anisopliae but only one from B. bassiana was documented by 1-DE native zymography combined with 2-D spot densitometry scans. Cationic PAGE native zymography separated two basic protease isozymes from Tw extract of M. anisopliae depending upon the pH of the incubation buffer. However, one activity band was identified from B. bassiana. Furthermore, only one activity band was apparent during 1st and 2nd Ww up to day 2 for both EPF. SDS PAGE (non-dissociating) zymogram of secreted protease isozymes from Tw of B. bassiana revealed three bands of Mr100, 60, and 36.3 kDa. The isozymes observed at day 2 and 3 had a Mrs of 35.4 and 25 kDa, and 24.7 and 20.3 kDa at day 4. The SDS PAGE zymograms for M. anisopliae indicated two isozymes of Mr 103 and 12 kDa, respectively. During the 1st Ww and incubation of spores at day 2 and 3, a 12 kDa band was observed. These results confirm the presence of diversity of proteases and their isozymes with unique molecular sizes.This thesis research discovered and characterized a diversity of proteins/enzymes not previously reported from any other fungi. A newly designed enzyme overlay membrane (EOM) technique revealed three isoforms of Pr1-like subtilisin from Tw of M. anisopliae (pI 8.1-9.7) and B. bassiana (pI 8.4-9.7). Conversely, only one isoform of Pr2-like trypsin was identified from M. anisopliae and no Pr2-like activity was observed from B. bassiana. Use of metalloprotease (MEP) inhibitors in conjunction with EOM analysis revealed their release during treatment in Tw. In M. anisopliae four activities (pI 4.4-7.5) of thermolysin-like MEP were observed. However, Tw of B. bassiana showed one activity band (pI 5.5). In addition, an isozyme of neutral MEP containing Zinc from M. anisopliae (pI 6.1) and one from B. bassiana (pI 6.5-7.6), respectively, was identified. MALDI-TOF and Q-TOF analysis revealed the presence of proteins similar to ROD 1, Ü- and â-glucanases, elastase, lipase 5 and galectin 7, which are important during the initial phase of germination and pathogenesis. In addition subtilisin (Pr1-like), trypsin (Pr2-like) and NAGase synthesis from the germinating conidia and mycelia under the supply of different carbon and nitrogen (C/ N) sources was studied. The regulation of the synthesis of cuticle-degrading enzymes (CDE) from germinating conidia and mycelia was hypothesized to be controlled through regulatory derepression and nutritional starvation. Pr1 and Pr2 are regulated in a different manner in conidia and mycelia. Both enzymes are regulated through a multiple control mode. It was concluded that C/ N repression occurs only when it is necessary for infective structures to establish a nutritional relationship with the host cuticular structures. In addition, C/ N sources have a significant effect upon pH modulation, ammonia production and protease secretion. Furthermore, the synthesis of Pr1 and Pr2 from germinating conidia was affected by the (inducer pH) pHi of the growth media. Growing mycelia of B. bassiana under acidic (4.0), neutral (7.0) and basic (11.0) pH conditions produce ammonia which modifies the pH thereby creating environments suitable for protease. Growth, morphology, radial extension rate and conidiation at different pHi revealed that both EPF modify the pH of growth medium effectively as opposed to the saprophytic fungus, Aspergillus nidulans. The presence of MEPs and Pr2-like trypsin suggests that these enzymes can act as a back up system for Pr1 to breach the cuticle and facilitate penetration before appressoria formation. The diversity of isozymes released from conidia suggests that the EPF are pre-adapted to pathogenic mode of life style, further contributing complexity to their interaction with host insects. Such isozymes can circumvent protease inhibitors present in the insect cuticle and the hemolymph. In addition, these isozymes may offer selective advantages in exploring new habitats (substrates) either as pathogen or saprophyte