186,849 research outputs found
Mass spectrometry approaches for the discovery and full primary structure analysis of peptides secreted by the amphibian skin
No full textMost, if not all, frog species are known to secrete a mixture of more or less toxic compounds from skin glands as defense mechanism. This skin secretion of amphibian constitutes a rich source of bioactive peptides with potential pharmaceutical applications. Research in this area includes peptidome (primary structure), transcriptome and bioassays analyses (Chapter 1). Although the complete genome of a frog species has been mapped, the analysis of frog peptides remains difficult. This is mainly because during evolution each frog species has gathered its own set of bioactive peptides encoded in its genes. In this thesis we focus on MS-based methods that help to characterize the peptidome of frog venoms. In Chapter 2 an overview is given of mass spectrometry methods and approaches to study frog skin secretions. Many of the frog skin peptides are post-translationally modified after their synthesis and mass spectrometry is an excellent tool to characterize them. This is illustrated in Chapter 2 for several frequently occurring post-translational modifications previously found on frog peptides. The skin secretion of the Chinese odorous frog Odorrana schmackeri had been studied before at the transcriptome level, leading to the discovery of full length open reading frames. The amino acid sequence of the mature peptide can be predicted from these open reading frames obtained by molecular cloning. After obtaining transcriptome results, mass spectrometry is the next logical step to proof the existence of the predicted peptides. Miniaturized nano liquid chromatography in combination with mass spectrometry is the preferred tool to do this as it requires minimal amounts of sample. In Chapter 3 a combination of transcriptome and peptidome analyses lead to the discovery and characterization of several members of the nigrocin peptide family found in the skin secretion of Odorrana schmackeri. The results from the MS measurements showed that all nigrocin peptides have a disulfide-bridge and from their peptide fragmentation spectra the complete amino acid sequence for each of the three nigrocins peptides could be confirmed. During these measurements of the nigrocin peptides, three other peptides caught our attention because of their a-typical peptide fragmentation behavior. Using both CID and ETD fragmentation analyses we could fully de novo sequence them and establish that they are O-linked glycosylated peptides (Chapter 4). All three peptides had the same primary structure and O-glycosylation at a Threonine residue. They differed in the monosaccharides composition (2, 3 and 4 units). Interestingly, the unmodified peptide (i.e. with no glycosylation) was not detected in the venom. Interestingly the very same primary structure has been found by molecular cloning in other Ranid frogs, however, obviously the glycosylation could not be (and was not) predicted from the cDNA/mRNA analysis. To our knowledge, this is the first example of a glycosylation as a PTM in amphibian skin secretion. The bioactivity of the peptide and the role of the glycosylations have yet to be determined. From the same analysis of the skin secretion of Odorrana schmackeri, yet two other peptides caught our interest. The majority of the peptides from Ranidae frogs contain a so called Rana box motif, which is a disulfide bridge in the C-terminal domain of the peptide. However, we discovered two relatively large peptides with a disulfide bridge at the N-terminal side, which is very uncommon for frog skin peptides. These peptides, with 34 residues, could be de novo sequenced using chemical treatments with DTT, iodoacetamide and bromoethylamine; high resolution CID and ETD fragmentation on full length peptides as well as on selected tryptic fragments; and labeling purified tryptic fragments with heavy and light dimethyl. The entire primary sequence, including Leu and Ile discriminations, was confirmed by Edman degradation. These two peptides share conservative motifs with calcitonin, adrenomedullin and calcitonin gene related peptides, but yet cannot be categorized in either of these existing peptide families. These unusual calcitonin-like peptide sequences (OsCLP1 and 2) present in the skin secretion of an anuran species may represent a novel peptide family within the calcitonin gene related peptide superfamily. Another type of venom which was studied during this research was that of the genus Phyllomedusa, which are known for their highly poisonous skin secretions. Specimens of the walking leaf frog, Phyllomedusa burmeisteri and Phyllomedusa rohdei were captured during hunting trips in Brazil. Not much was known yet about the peptides present in their toxic secretions. Their skin secretions were collected by a non-invasive method and afterwards all specimen were immediately released in their natural surroundings. The skin secretions of both frogs were used to optimize our MS-based methods and approaches and this has led to the discovery of several novel peptides. In Chapter 6 a generic method is presented to detect peptides with intra- or an inter-molecular disulfide bonds prior to MS based sequencing of their primairy structure. The method is based upon the comparative analysis by LC-MS of the untreated crude venom and the crude venom in which all S-S bonds are broken with a reducing agent, such as dithiotreitol. The LC-MS data are converted into a so called peptide display, in which the retention time is plotted against the mass-over-charge ratio. By comparing the peptide display of both analyses, peptides with a disulfide bond(s) are easily recognized, because their mass increases by 2 Da per broken disulfide bond, and their retention times usually also slightly shift. Beside the two Phyllomedusa species, 3 other types of frog venoms were subjected to this method and it was observed that each venom shows different profiles. O. schmackeri, for example, is highly rich in intramolecular disulfide bonds. The skin secretion of P. burmeisteri and P. rohdei contain several large peptides containing 3 S-S bonds, Kassina senegalensis contains overall a less complex peptide content and only a few peptides with a S-S bridge, whereas Bombina variegata venom yielded several peptides with single or double S-S bonds. Inter-molecular disulfide bonds were also observed by this approach in the venom of P. burmeisteri, i.e., the distinctin homo- and heterodimers. In Chapter 7 we describe studies involving the shotgun cloning of the distinctin chains from the skin secretion. cDNA cloning surprisingly revealed that the chains A and B are encoded by separate mRNAs. Oxidation experiments with the synthetic chains did not show any preferential formation over the dimerization, suggesting either that there is an alternative mechanism for dimer formation in vivo or that synthetic chains have different behavior than native peptides. The P. burmeisteri and P. rohdei peptides described in Chapter 6 that contain 3 disulfide bonds as determined by the 2-D differential peptide display method are further characterized in Chapter 8. Several MS-based approach were used to de novo sequence these large 6 kDa peptides, including complementary CID and ETD peptide fragmentation and high resolution orbitrap detection. These peptides showed several conservative motifs of the Kazal peptides, a group of protease inhibitors. An interesting observation made during these analyses is that peptides modified with bromoethylamine show an much better ETD fragmentation pattern compared to the same peptides modified with iodoacetamide, which is the most commonly used reagent to block free cysteines in proteomics. Another important issue to mention is that for this, and all the other MS based methods described in this thesis, only little amount of material was used, which is crucial for frog skin researches on limited samples which are difficult to acquire. Moreover, the methodologies showed in this thesis can be applied to other peptidomic samples. Finally in Chapter 9 all important findings done during this research are discussed, several concluding remarks are given and a future outlook is presented.BiotechnologyApplied Science
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Peptide hormone immunocytochemistry in insects
No full textUsing a number of specific polyclonal and monoclonal
(Mab) antisera antibodies to vertebrate peptide hormones
related to the gastro-entero-pancreatic system,
positive immunohistochemical results were obtained
in the American cockroach, Periplaneta americana (L.).
Neurosecretory neuron-like cells and their processes
in the brain and/or nerve terminals in the corpus
cardiacum (an important insect neurohaemal organ)
were observed to be selectively labelled by antibodies
to vasoactive intestinal polypeptide and peptide histidine
isoleucine 1 (labelling distinct materials), gastrin
and cholecystokinin 2 (also resulting in a different
distribution of the immunoreaction), bombesin/gastrin
releasing peptide I (two different antisera giving
entirely different staining patterns), neurotensin 3,
insulin-like growth factor I (Mab), insulin, glucagon 3,
somatostatin, pancreatic polypeptide and substance P
(Mab), the latter two being published earlier [Verhaert
et al., Brain Res. 348, 331-8 (1985); Verhaert & De Loof,
Histochemistry 83, 501-7 (1985), respectively]. Moreover,
except for the PHI antiserum, which only stained
some intestinal nerves, antibodies to all these hormones
specifically marked both cells of the paraneuron
(endocrine) type in the midgut wall and nerve
fibres terminating on the alimentary epithelium.
These data are in favour of the theory that in insects as
well as in vertebrates, peptide-like chemicals may exist
having a dual localization, i.e. centrally, in the brain as
well as peripherally, in the gut. Preliminary experiments
using radiolabelled vertebrate glucagon and
insulin cDNAs in in situ hybrization histochemistry
indicate that one has to be very cautious in interpreting
(RNA dependent) hybridization results obtained
with heterologous probes. A general (non-specific?)
distribution of radiolabelling was observed in the
cockroach brain cells, in identical conditions of stringency
(i.e. 50% formamide, 37°C, 2xSSC) which allow a
(specific) marking of the relevant cells of vertebrate
pancreatic tissue.status: Publishe
Editorial: The EuPA Company Club: Status, report, developments and perspectives
No full textThe high-temperature phase diagram of the UO2–ThO2 system has been experimentally revisited in the present study for the first time since 1970, using a laser heating approach combined with fast pyrometry in a thermal arrest method. The melting/solidification temperature, which is of fundamental information for a reactor design was studied here. It was found that low addition of ThO2 to UO2 would result in a slight decrease of the solidification temperature. A minimum was found at 3098 K around a composition of 5 mol% ThO2. The solid/liquid transition temperature was then observed to increase again with increasing ThO2 fraction. The literature value of pure ThO2 (around 3630 K) was well reproduced here. Important experimental difficulties, stemming from the high temperatures reached during the measurements, as well as a complete investigation with electron microscopy, Raman spectroscopy and powder X-ray diffraction, are extensively discussed. These results show the importance of the high-temperature oxygen chemistry in this actinide oxide compound.BT/BiotechnologyApplied Science
sj-pdf-1-opp-10.1177_10781552231167824 - Supplemental material for Immunosuppressive therapy management in cancer patients with autoimmune diseases treated with immune checkpoint inhibitors: A case series and systematic literature review
No full textSupplemental material, sj-pdf-1-opp-10.1177_10781552231167824 for Immunosuppressive therapy management in cancer patients with autoimmune diseases treated with immune checkpoint inhibitors: A case series and systematic literature review by Stephanie C.M. Wuyts, Charlotte A.H. Cappelle, Marthe Verhaert, Bert Bravenboer and Sandrine Aspeslagh in Journal of Oncology Pharmacy Practice</p
Appropriate Similarity Measures for Author Cocitation Analysis
No full textWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
No full textWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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