Inhibin secretion in women with the polycystic ovary syndrome before and after treatment with progesterone

Objectives  

It has been suggested that inhibin secretion is altered in women with the polycystic ovary syndrome (PCOS). However, the contribution of a preceding luteal phase has not been taken into account. The aim of the present study was to investigate whether progesterone in the context of a simulated luteal phase affects basal and FSH-induced inhibin secretion in women with PCOS and elevated LH.     

Proteomics studies of childhood pilocytic astrocytoma

Childhood pilocytic astrocytoma is the most frequent brain tumor affecting children. Proteomics analysis is currently considered a powerful tool for global evaluation of protein expression and has been widely applied in the field of cancer research. In the present study, a series of proteomics, genomics, and bioinformatics approaches were employed to identify, classify and characterize the proteome content of low-grade brain tumors as it appears in early childhood. Through bioinformatics database construction, protein profiles generated from pathological tissue samples were compared against profiles of normal brain tissues. Additionally, experiments of comparative genomic hybridization arrays were employed to monitor for genetic aberrations and sustain the interpretation and evaluation of the proteomic data. The current study confirms the dominance of MAPK pathway for the childhood pilocytic astrocytoma occurrence and novel findings regarding the ERK-2 expression are reported.     

Pressurized Pepsin Digestion in Proteomics: AN AUTOMATABLE ALTERNATIVE TO TRYPSIN FOR INTEGRATED TOP-DOWN BOTTOM-UP PROTEOMICS*

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.In-depth characterization and quantitation of protein isoforms, including post-translationally modified proteins, are challenging goals of contemporary proteomics. Traditionally, top-down (1, 2) and bottom-up (3, 4) proteomics have been two distinct analytical paths for liquid-based proteomics analysis. Top-down proteomics is the mass spectrometry (MS)-based characterization of intact proteins, whereas bottom-up proteomics requires a chemical or enzymatic proteolytic digestion of all proteins into peptides prior to MS analysis. Both strategies have their own strengths and challenges and can be thought of as complementary rather than competing analytical techniques.In a top-down proteomics approach, proteins are usually separated by one- or two-dimensional liquid chromatography (LC) and identified using high performance MS (5, 6). This approach is very attractive because it allows the identification of protein isoforms arising from various amino acid modifications, genetic variants (e.g. single nucleotide polymorphisms), mRNA splice variants, and multisite modifications (7) (e.g. specific histone modifications) as well as characterization of proteolytic processing events. However, there are several challenges that have limited the broad application of the approach. Typically, intact proteins are less soluble than their peptide complement, which effectively results in greater losses during various stages of sample handling (i.e. limited sensitivity). Similarly, proteins above ∼40–50 kDa in size are more difficult to ionize, detect, and dissociate in most high throughput MS work flows. Additionally, major challenges associated with MS data interpretation and sensitivity, especially for higher molecular mass proteins (>100 kDa) and highly hydrophobic proteins (e.g. integral membrane proteins), remain largely unsolved, thus limiting the applicability of top-down proteomics on a large scale.Bottom-up proteomics approaches have broad application because peptides are easier to separate and analyze via LC coupled with tandem mass spectrometry (MS/MS), offering a basis for more comprehensive protein identification. As this method relies on protein digestion (which produces multiple peptides for each protein), the sample complexity can become exceedingly large, requiring several dimensions of chromatographic separations (e.g. strong cation exchange and/or high pH reversed phase) prior to the final LC separation (typically reversed phase (RP)¹ C₁₈), which is oftentimes directly coupled with the mass spectrometer (3, 8). In general, the bottom-up analysis rarely achieves 100% sequence coverage of the original proteins, which can result in an incorrect/incomplete assessment of protein isoforms and combinatorial PTMs. Additionally, the digested peptides are not detected with uniform efficiency, which challenges and distorts protein quantification efforts.Because the data obtained from top-down and bottom-up work flows are complementary, several attempts have been made to integrate the two strategies (9, 10). Typically, these efforts have utilized extensive fractionation of the intact protein separation followed by bottom-up analysis of the collected fractions. Results so far have encouraged us to consider on-line digestion methods for integrating top-down and bottom-up proteomics in a higher throughput fashion. Such an on-line digestion approach would not only benefit in terms of higher sample throughput and improved overall sensitivity but would also allow a better correlation between the observed intact protein and its peptide digestion products, greatly aiding data analysis and protein characterization efforts.So far, however, none of the on-line integrated methods have proven robust enough for routine high throughput analyses. One of the reasons for this limited success relates to the choice of the proteolytic enzyme used for the bottom-up segment. Trypsin is by far the most widely used enzyme for proteome analyses because it is affordable (relative to other proteases), it has been well characterized for proteome research, and it offers a nice array of detectable peptides due to a fairly even distribution of lysines and arginines across most proteins. However, protein/peptide RPLC separations (optimal at low pH) are fundamentally incompatible with on-line trypsin digestion (optimal at pH ∼ 8) (11, 12). Therefore, on-line coupling of trypsin digestion and RPLC separations is fraught with technological challenges, and proposed solutions (12) have not proven to be robust enough for integration into demanding high throughput platforms.Our approach to this challenge was to investigate alternative proteases that may be more compatible with automated on-line digestion, peptide separation, and MS detection. Pepsin, which is acid-compatible (i.e. it acts in the stomach to initially aid in the digestion of food) (13), is a particularly promising candidate. This protease has previously been successfully used for the targeted analyses of protein complexes, hydrogen/deuterium exchange experiments (14, 15), and characterization of biopharmaceuticals (16, 17). Generally, pepsin preferentially cleaves the peptide bond located on the N-terminal side of hydrophobic amino acids, such as leucine and phenylalanine, although with less specificity than the preferential cleavage observed for trypsin at arginine and lysine. The compatibility of pepsin with typical LC-MS operation makes it an ideal choice for the development of novel approaches combining protein digestion, protein/peptide separation, and MS-based protein/peptide identification.To develop an automated system capable of simultaneously capturing top-down and bottom-up data, enzyme kinetics of the chosen protease must be extremely fast (because one cannot wait hours as is typical when performing off-line proteolysis). Another requirement is the use of immobilized enzyme or a low enough concentration of the enzyme such that autolysis products do not obscure the detection of substrate peptides. The latter was a concern when using pepsin because prior hydrogen/deuterium exchange experiments used enzyme:substrate ratios up to 1:2 (18, 19). To test whether or not such a large concentration of pepsin was necessary, we performed pepsin digestion at ratios of 1:20. Many alternative energy inputs into the system were considered for speeding up the digestion. For instance, it has been shown that an input of ultrasonic energy could accelerate the reaction rate of a typical trypsin digestion while using small amounts of a protease (20). Because ultrasonic energy results in an increase of temperature and microenvironments of high pressure, it has been hypothesized that the higher temperature was the component responsible for the enhanced enzyme activity (21). López-Ferrer et al. (22, 23), however, have demonstrated that application of higher pressure with incorporation of a Barocycler alone can make trypsin display faster enzyme kinetics. This phenomenon can easily be integrated with an LC separation (which already operates at elevated pressure) to enable an automatable ultrarapid on-line digestion LC-MS proteomics platform. Herein, we refer to this platform as the fast on-line digestion system (FOLDS) (23). Although FOLDS has been described before using trypsin, here the system is characterized with pepsin, and the results obtained are compared with results attainable with trypsin. Like trypsin, pepsin produced efficient protein digestion in just a few minutes when placed under pressure. Because of the natural maximal activity of pepsin at low pH, the FOLDS can be incorporated with a RePlay (Advion Biosciences, Ithaca, NY) system, and this powerful combination is what ultimately makes the integration of top-down and bottom-up proteomics analyses possible. The integrated analysis begins with a chromatographic separation of intact proteins. The separated proteins are then split into two streams. One stream proceeds directly to the mass spectrometer for MS and/or tandem MS analysis. The second stream is split into a long capillary where the chromatographic separation of the proteins is maintained, but their arrival to the mass spectrometer for detection is delayed. This is in essence the concept of RePlay (24, 25). Herein, we have taken the RePlay a step further by implementing our FOLDS technology into the second split delayed stream of proteins. While these delayed proteins travel down the long and narrow capillary, we exposed them to pepsin where, in combination with the pressure, the proteins are quickly and reproducibly digested. These peptide fragments are subsequently subjected to MS and/or tandem MS analysis. The FOLDS RePlay system allows the rapid and robust incorporation of the integrated top-down bottom-up proteomics work flow with the ability to not only identify proteins but also to sequence multisite/combinatorial PTMs because all detected peptides (from the FOLDS analysis) are confined to the original chromatographic peak of the protein they were derived from. The analysis of protein mixtures using this integrated strategy reduces the total amount of samples required to obtain both the top-down and bottom-up data, increases throughput, and improves protein sequence coverage.     

Functionality of natural killer cells from end-stage cancer patients exposed to coherent electromagnetic fields

The main objective of our study is to investigate whether an enhancement of the immune system in end-stage cancer patients is achieved by exposure to coherent electromagnetic fields. For this reason, 15 end-stage cancer patients were exposed at low intensity, coherent electromagnetic fields at radiofrequencies ranging from 600?kHz-729?Hz, for 8?h/day, 6 days/week for 4 weeks. NKs number and cytotoxicity of NK T-lymphocytes versus K562 cancer cell line were estimated by flow cytometry, before and after exposure. Data showed that the exposure of the end-stage cancer patients to the coherent electromagnetic fields resulted in a significant increase of the number and the cytotoxicity of the NK T-lymphocytes against cancer cells, in all patients. Exposure to coherent EMFs at radiofrequencies increases the number and cytotoxicity of NK T-lymphocytes, which may contribute to the improvement of cancer patients' status.     

Lipid synthesized by micro‐algae grown in laboratory‐ and industrial‐scale bioreactors

Tetraselmis sp. and Nannochloropsis oculata, cultivated in industrial‐scale bioreactors, produced 2.33 and 2.44% w/w lipid (calculated as the sum of fatty acid methyl esters) in dry biomass, respectively. These lipids contained higher amounts of neutral lipids and glycolipids plus sphingolipids, than phospholipids. Lipids of Tetraselmis sp. were characterized by the presence of eicosapentaenoic acid (that was located mainly in phospholipids), and octadecatetraenoic acid (that was equally distributed among lipid fractions), while these fatty acids were completely absent in N. oculata lipids. Additionally, lipids produced by 16 newly isolated strains from Greek aquatic environments (cultivated in flask reactors) were studied. The highest percentage of lipids was found in Prorocentrum triestinum (3.69% w/w) while the lowest in Prymnesium parvum (0.47% w/w). Several strains produced lipids rich in eicosapentaenoic and docosahexaenoic acids. For instance, docosahexaenoic acid was found in high percentages in lipids of Amphidinium sp. S1, P. parvum, Prorocentrum minimum and P. triestinum, while lipids produced by Asterionella sp. (?) S2 contained eicosapentaenoic acid in high concentration. These lipids, containing ω‐3‐long‐chain polyunsaturated fatty acids, have important applications in the food and pharmaceutical industries and in aquaculture.     

Morphing of the phylogeographic lineages of the Balkan alpine newts (Ichthyosaura alpestris, Caudata, Salamandridae): in situ morphological diversification

Numerous alpine newt (Ichthyosaura alpestris) populations from the Balkans, representing all the previously established phylogeographic lineages, were studied for variations in various morphological characteristics (body size and shape, skull qualitative traits and number of trunk vertebrae). Here, we present a decoupling of morphological and mtDNA phylogeographic substructuring in the alpine newt on the Balkan Peninsula. In sharp contrast to other European newts (Triturus spp., Lissotriton spp.), the vast majority of morphological variation in the alpine newt is concentrated at the population level indicating an in situ morphological diversification. We found that the rate of morphological change is similar to the rate of mtDNA change. We hypothesize that the alpine newts are characterized by non-adaptive morphological evolution.     

Complete genome sequence of Bacteroides helcogenes type strain (P 36-108)

Bacteroides helcogenes Benno et al. 1983 is of interest because of its isolated phylogenetic location and, although it has been found in pig feces and is known to be pathogenic for pigs, occurrence of this bacterium is rare and it does not cause significant damage in intensive animal husbandry. The genome of B. helcogenes P 36-108(T) is already the fifth completed and published type strain genome from the genus Bacteroides in the family Bacteroidaceae. The 3,998,906 bp long genome with its 3,353 protein-coding and 83 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of "Enterobacter lignolyticus" SCF1

In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated "Enterobacter lignolyticus" SCF1 on minimal media with alkali lignin as the sole source of carbon. This organism was isolated anaerobically from tropical forest soils collected from the Short Cloud Forest site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are net methane producers. Because of its ability to grow on lignin anaerobically, we sequenced the genome. The genome of "E. lignolyticus" SCF1 is 4.81 Mbp with no detected plasmids, and includes a relatively small arsenal of lignocellulolytic carbohydrate active enzymes. Lignin degradation was observed in culture, and the genome revealed two putative laccases, a putative peroxidase, and a complete 4-hydroxyphenylacetate degradation pathway encoded in a single gene cluster.     

Complete genome sequence of Thermomonospora curvata type strain (B9)

Thermomonospora curvata Henssen 1957 is the type species of the genus Thermomonospora. This genus is of interest because members of this clade are sources of new antibiotics, enzymes, and products with pharmacological activity. In addition, members of this genus participate in the active degradation of cellulose. This is the first complete genome sequence of a member of the family Thermomonosporaceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,639,016 bp long genome with its 4,985 protein-coding and 76 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Hydrogenobacter thermophilus type strain (TK-6)

Hydrogenobacter thermophilus Kawasumi et al. 1984 is the type species of the genus Hydrogenobacter. H. thermophilus was the first obligate autotrophic organism reported among aerobic hydrogen-oxidizing bacteria. Strain TK-6(T) is of interest because of the unusually efficient hydrogen-oxidizing ability of this strain, which results in a faster generation time compared to other autotrophs. It is also able to grow anaerobically using nitrate as an electron acceptor when molecular hydrogen is used as the energy source, and able to aerobically fix CO(2)via the reductive tricarboxylic acid cycle. This is the fifth completed genome sequence in the family Aquificaceae, and the second genome sequence determined from a strain derived from the original isolate. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,742,932 bp long genome with its 1,899 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.