AcT-2: A Novel Myotropic and Antimicrobial Type 2 Tryptophyllin from the Skin Secretion of the Central American Red-Eyed Leaf Frog, Agalychnis callidryas

Tryptophyllins are a diverse family of amphibian peptides originally found in extracts of phyllomedusine frog skin by chemical means. Their biological activities remain obscure. Here we describe the isolation and preliminary pharmacological characterization of a novel type 2 tryptophyllin, named AcT-2, from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas. The peptide was initially identified during smooth muscle pharmacological screening of skin secretion HPLC fractions and the unique primary structure—GMRPPWF-NH2—was established by both Edman degradation and electrospray MS/MS fragmentation sequencing. A. cDNA encoding the biosynthetic precursor of AcT-2 was successfully cloned from a skin secretion-derived cDNA library by means of RACE PCR and this contained an open-reading frame consisting of 62 amino acid residues with a single AcT-2 encoding sequence located towards the C-terminus. A synthetic replicate of AcT-2 was found to relax arterial smooth muscle (EC50 = 5.1 nM) and to contract rat urinary bladder smooth muscle (EC50 = 9.3 μM). The peptide could also inhibit the growth of the microorganisms, Staphylococcus aureus, (MIC = 256 mg/L) Escherichia coli (MIC = 512 mg/L), and Candida albicans (128 mg/L). AcT-2 is thus the first amphibian skin tryptophyllin found to possess both myotropic and antimicrobial activities.

Ranakinestatin-PPF from the Skin Secretion of the Fukien Gold-Striped Pond Frog, Pelophylax plancyi fukienensis: A Prototype of a Novel Class of Bradykinin B2 Receptor Antagonist Peptide from Ranid Frogs

The defensive skin secretions of many amphibians are a rich source of bradykinins and bradykinin-related peptides (BRPs). Members of this peptide group are also common components of reptile and arthropod venoms due to their multiple biological functions that include induction of pain, effects on many smooth muscle types, and lowering systemic blood pressure. While most BRPs are bradykinin receptor agonists, some have curiously been found to be exquisite antagonists, such as the maximakinin gene-related peptide, kinestatin—a specific bradykinin B₂-receptor antagonist from the skin of the giant fire-bellied toad, Bombina maxima. Here, we describe the identification, structural and functional characterization of a heptadecapeptide (DYTIRTRLHQGLSRKIV), named ranakinestatin-PPF, from the skin of the Chinese ranid frog, Pelophylax plancyi fukienensis, representing a prototype of a novel class of bradykinin B₂-receptor specific antagonist. Using a preconstricted preparation of rat tail arterial smooth muscle, a single dose of 10⁻⁶ M of the peptide effectively inhibited the dose-dependent relaxation effect of bradykinin between 10⁻¹¹ M and 10⁻⁵ M and subsequently, this effect was pharmacologically-characterized using specific bradykinin B₁- (desArg-HOE140) and B₂-receptor (HOE140) antagonists; the data from which demonstrated that the antagonism of the novel peptide was mediated through B₂-receptors. Ranakinestatin—PPF—thus represents a prototype of an amphibian skin peptide family that functions as a bradykinin B₂-receptor antagonist herein demonstrated using mammalian vascular smooth muscle.

Ornithokinin (avian bradykinin) from the skin of the Chinese bamboo odorous frog, Odorrana versabilis

One of the most widespread and abundant families of pharmacologically active peptides in amphibian defensive skin secretions is the bradykinins and related peptides. Despite retaining certain primary structural attributes that assign them to this peptide family, bradykinins and related peptides are unique among amphibian skin peptides in that they exhibit a wide range of primary structural variations, post-translational modifications and/or N-terminal or C-terminal extensions. Initially it was believed that their high degree of primary structural heterogeneity was reflective of random gene mutations within species, but latterly, there is an increasing body of evidence that the spectrum of structural modifications found within this peptide family is reflective of the vertebrate predator spectrum of individual species. Here we report the discovery of ornithokinin (avian bradykinin – Thr6, Leu8-bradykinin) in the skin secretion of the Chinese bamboo odorous frog, Odorrana versabilis. Molecular cloning of its biosynthetic precursor-encoding cDNA from a skin secretion-derived cDNA library revealed a deduced open-reading frame of 86 amino acid residues, encoding a single copy of ornithokinin towards its C-terminus. The domain architecture of this ornithokinin precursor protein was consistent with that of a typical amphibian skin peptide and quite different to that of the ornithokininogen from chicken plasma. Ornithokinin was reported to induce hypotension in the chicken and to contract the chicken oviduct but to have no obvious effect on the rat uterus. However, in this study, synthetic ornithokinin was found to contract the rat ileum (EC50 = 539 nM) and to increase contraction frequency in the rat uterus (EC50 = 1.87 μM).

Molecular Cloning of a Novel Tryptophyllin Peptide from the Skin of the Orange-Legged Monkey Frog, Phyllomedusa hypochondrialis

Tryptophyllins are a group of small (4–14 amino acids), heterogenous peptides, mostly from the skins of hylid frogs from the genera, Phyllomedusa and Litoria. To date, more than forty TPHs have been discovered in species from these two genera. Here, we describe the identification of a novel tryptophyllin type 3 peptide, PhT-3, from the extracts of skin of the orange-legged monkey frog, Phyllomedusa hypochondrialis, and molecular cloning of its precursor-encoding cDNA from a cDNA library constructed from the same skin sample. Full primary structural characterization was achieved using a combination of direct Edman degradation, mass spectrometry and deduction from cloned skin-derived cDNA. The open-reading frame of the precursor cDNA was found to consist of 63 amino acid residues. The mature peptide arising from this precursor contains a post-translationally modified N-terminal pyroglutamate (pGlu) residue, formed from acid-mediated cyclization of an N-terminal Gln (Q) residue, and with the structure: pGlu-Asp-Lys-Pro-Phe-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met. Pharmacological assessment of a synthetic replicate of this peptide on phenylephrine preconstricted rat tail artery segments, revealed a reduction in relaxation induced by bradykinin. PhT-3 was also found to mediate antiproliferative effects on human prostate cancer cell lines.

Tackling Invasive Alien Species in Europe: the Top 20 Issues

Globally, Invasive Alien Species (IAS) are considered to be one of the major threats to native biodiversity, with the World Conservation Union (IUCN) citing their impacts as ?immense, insidious, and usually irreversible?. It is estimated that 11% of the c. 12,000 alien species in Europe are invasive, causing environmental, economic and social damage; and it is reasonable to expect that the rate of biological invasions into Europe will increase in the coming years. In order to assess the current position regarding IAS in Europe and to determine the issues that were deemed to be most important or critical regarding these damaging species, the international Freshwater Invasives - Networking for Strategy (FINS) conference was convened in Ireland in April 2013. Delegates from throughout Europe and invited speakers from around the world were brought together for the conference. These comprised academics, applied scientists, policy makers, politicians, practitioners and representative stakeholder groups. A horizon scanning and issue prioritization approach was used by in excess of 100 expert delegates in a workshop setting to elucidate the Top 20 IAS issues in Europe. These issues do not focus solely on freshwater habitats and taxa but relate also to marine and terrestrial situations. The Top 20 issues that resulted represent a tool for IAS management and should also be used to support policy makers as they prepare European IAS legislation.

Feleucins: Novel Bombinin Precursor-Encoded Nonapeptide Amides from the Skin Secretion of Bombina variegata

The first amphibian skin antimicrobial peptide (AMP) to be identified was named bombinin, reflecting its origin from the skin of the European yellow-bellied toad (Bombina variegata). Bombinins and their related peptides, the bombinin Hs, were subsequently reported from other bombinid toads. Molecular cloning of bombinin-encoding cDNAs from skin found that bombinins and bombinin Hs were coencoded on the same precursor proteins. Here, we report the molecular cloning of two novel cDNAs from a skin secretion-derived cDNA library of B. variegata whose open-reading frames each encode a novel bombinin (GIGGALLNVGKVALKGLAKGLAEHFANamide) and a C-terminally located single copy of a novel nonapeptide (FLGLLGGLLamide or FLGLIGSLLamide). These novel nonapeptides were named feleucin-BV1 and feleucin-BV2, respectively. The novel bombinin exhibited 89% identity to homologues from the toads, B. microdeladigitora and B. maxima. The feleucins exhibited no identity with any amphibian AMP archived in databases. Synthetic feleucins exhibited a weak activity against Staphylococcus aureus (128–256 mg/L) but feleucin-BV1 exhibited a synergistic action with the novel bombinin. The present report clearly demonstrates that the skin secretions of bombinid toads continue to represent a source of peptides of novel structure that could provide templates for the design of therapeutics.

Balteatide: A Novel Antimicrobial Decapeptide from the Skin Secretion of the Purple-Sided Leaf Frog, Phyllomedusa baltea

The skin secretions of Neotropical phyllomedusine leaf frogs have proven to be a rich source of biologically-active peptides, including antimicrobials. The major families of antimicrobial peptides (AMPs) reported are the dermaseptins and phylloseptins and the minor families, the dermatoxins, phylloxins, plasticins, distinctins and the medusins. Here, we report a novel AMP of 10 amino acid residues (LRPAILVRIKamide), named balteatide, from the skin secretion of wild Peruvian purple-sided leaf frogs, Phyllomedusa baltea. Balteatide was found to exhibit a 90% sequence identity with sauvatide, a potent myotropic peptide from the skin secretion of Phyllomedusa sauvagei. However, despite both peptides exhibiting only a single amino acid difference (I/T at position 9), sauvatide is devoid of antimicrobial activity and balteatide is devoid of myotropic activity. Balteatide was found to have differential activity against the Gram-positive bacterium, Staphylococcus aureus, the Gram-negative bacterium, Escherichia coli and the yeast, Candida albicans, and unusually for phyllomedusine frog skin AMPs, was most potent (MIC 32 mg/L) against the yeast. Balteatide was also devoid of haemolytic activity up to concentrations of 512 mg/L. Phyllomedusine frog skin secretions thus continue to provide novel AMPs, some of which may provide templates for the rational design of new classes of anti-infective therapeutics.

Palynological perspectives on vegetation survey: a critical step for model-based reconstruction of Quaternary land cover

1. Quantitative reconstruction of past vegetation distribution and abundance from sedimentary pollen records provides an important baseline for understanding long term ecosystem dynamics and for the calibration of earth system process models such as regional-scale climate models, widely used to predict future environmental change. Most current approaches assume that the amount of pollen produced by each vegetation type, usually expressed as a relative pollen productivity term, is constant in space and time.
2. Estimates of relative pollen productivity can be extracted from extended R-value analysis (Parsons and Prentice, 1981) using comparisons between pollen assemblages deposited into sedimentary contexts, such as moss polsters, and measurements of the present day vegetation cover around the sampled location. Vegetation survey method has been shown to have a profound effect on estimates of model parameters (Bunting and Hjelle, 2010), therefore a standard method is an essential pre-requisite for testing some of the key assumptions of pollen-based reconstruction of past vegetation; such as the assumption that relative pollen productivity is effectively constant in space and time within a region or biome.
3. This paper systematically reviews the assumptions and methodology underlying current models of pollen dispersal and deposition, and thereby identifies the key characteristics of an effective vegetation survey method for estimating relative pollen productivity in a range of landscape contexts.
4. It then presents the methodology used in a current research project, developed during a practitioner workshop. The method selected is pragmatic, designed to be replicable by different research groups, usable in a wide range of habitats, and requiring minimum effort to collect adequate data for model calibration rather than representing some ideal or required approach. Using this common methodology will allow project members to collect multiple measurements of relative pollen productivity for major plant taxa from several northern European locations in order to test the assumption of uniformity of these values within the climatic range of the main taxa recorded in pollen records from the region.

The Holocene vegetation cover of Britain and Ireland: overcoming problems of scale and discerning patterns of openness

The vegetation of Europe has undergone substantial changes during the course of the Holocene epoch, resulting from range expansion of plants following climate amelioration, competition between taxa and disturbance through anthropogenic activities. Much of the detail of this pattern is understood from
decades of pollen analytical work across Europe, and this understanding has been used to address questions relating to vegetation-climate feedback, biogeography and human impact. Recent advances in modelling the relationship between pollen and vegetation now make it possible to transform pollen
proportions into estimates of vegetation cover at both regional and local spatial scales, using the Landscape Reconstruction Algorithm (LRA), i.e. the REVEALS (Regional Estimates of VEgetation Abundance from Large Sites) and the LOVE (LOcal VEgetation) models. This paper presents the compilation and analysis of 73 pollen stratigraphies from the British Isles, to assess the application of the LRA and describe the pattern of landscape/woodland openness (i.e. the cover of low herb and bushy vegetation) through the Holocene. The results show that multiple small sites can be used as an effective replacement for a single large site for the reconstruction of regional vegetation cover. The REVEALS vegetation estimates imply that the British Isles had a greater degree of landscape/woodland openness at the regional scale than areas on the European mainland. There is considerable spatial bias in the British Isles dataset towards wetland areas and uplands, which may explain higher estimates of landscape openness compared with Europe. Where multiple estimates of regional vegetation are available from within the same region inter-regional differences are greater than intra-regional differences, supporting the use of the REVEALS model to the estimation of regional vegetation from pollen data.

Predictors and dynamics of postpartum relapses in women with multiple sclerosis

Several studies have shown that pregnancy reduces multiple sclerosis (MS) relapses, which increase in the early postpartum period. Postpartum relapse risk has been predicted by pre-pregnancy disease activity in some studies.

Feleucin-BO1: A Novel Antimicrobial Non-Apeptide Amide from the Skin Secretion of the Toad, Bombina orientalis, and Design of a Potent Broad-Spectrum Synthetic Analogue, Feleucin-K3

Feleucins-BV1 and -BV2 are recently-described prototypes of a novel antimicrobial nonapeptide (AMP) family identified in the skin secretion of the bombinid toad, Bombina variegata. They are encoded on different precursors that also encode a novel bombinin. Here we describe the identification of feleucin-BO1 (FLGLLGSLLamide) which is co-encoded with a different novel bombinin, named feleucin precursor-associated bombinin (FPA-bombinin-BO), from the skin secretion of Bombina orientalis. Synthetic feleucin-BO1 displayed activity against a reference Gram-positive bacterium. Staphylococcus aureus (MIC 34 μM) but was inactive (> 250 μM) against the Gram-negative bacterium, Escherichia coli, and the yeast, Candida albicans. This pattern of activity was similar to that of the prototypes. Design and synthesis of a cationicity-enhanced analogue, feleucin-K3 (F-K3), in which the amino acid residues at positions 3 (G), 6 (G) and 7 (S) of feleucin-BO1 were substituted with Lys (K) residues, resulted in a peptide with significantly-enhanced potency and spectrum of activity. The MICs of F-K3 against the reference microorganisms were 7 μM (S. aureus), 14 μM (E. coli) and 7 μM (C. albicans). These data indicate that the skin secretions of amphibians can continue to provide novel peptide templates for the rational design of analogues with possible therapeutic utility.

Cationicity-Enhanced Analogues of the Antimicrobial Peptides, AcrAP1 and AcrAP2, from the Venom of the Scorpion, Androctonus crassicauda, Display Potent Growth Modulation Effects on Human Cancer Cell Lines

The non disulphide-bridged peptides (NDBPs) of scorpion venoms are attracting increased interest due to their structural heterogeneity and broad spectrum of biological activities. Here, two novel peptides, named AcrAP1 and AcrAP2, have been identified in the lyophilised venom of the Arabian scorpion, Androctonus crassicauda, through “shotgun” molecular cloning of their biosynthetic precursor-encoding cDNAs. The respective mature peptides, predicted from these cloned cDNAs, were subsequently isolated from the same venom sample using reverse phase HPLC and their identities were confirmed by use of mass spectrometric techniques. Both were found to belong to a family of highly-conserved scorpion venom antimicrobial peptides - a finding confirmed through the biological investigation of synthetic replicates. Analogues of both peptides designed for enhanced cationicity, displayed enhanced potency and spectra of antimicrobial activity but, unlike the native peptides, these also displayed potent growth modulation effects on a range of human cancer cell lines. Thus natural peptide templates from venom peptidomes can provide the basis for rational analogue design to improve both biological potency and spectrum of action. The diversity of such templates from such natural sources undoubtedly provides the pharmaceutical industry with unique lead compounds for drug discovery.

A Novel Bradykinin-Related Dodecapeptide (RVALPPGFTPLR) from the Skin Secretion of the Fujian Large-Headed Frog (Limnonectes fujianensis) Exhibiting Unusual Structural and Functional Features

Bradykinin-related peptides (BRPs) are significant components of the defensive skin secretions of many anuran amphibians, and these secretions represent the source of the most diverse spectrum of such peptides so far encountered in nature. Of the many families of bioactive peptides that have been identified from this source, the BRPs uniquely appear to represent homologues of counterparts that have specific distributions and receptor targets within discrete vertebrate taxa, ranging from fishes through mammals. Their broad spectra of actions, including pain and inflammation induction and smooth muscle effects, make these peptides ideal weapons in predator deterrence. Here, we describe a novel 12-mer BRP (RVALPPGFTPLR-RVAL-(L1, T6, L8)-bradykinin) from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis). The C-terminal 9 residues of this BRP (-LPPGFTPLR) exhibit three amino acid substitutions (L/R at Position 1, T/S at Position 6 and L/F at Position 8) when compared to canonical mammalian bradykinin (BK), but are identical to the kinin sequence present within the cloned kininogen-2 from the Chinese soft-shelled turtle (Pelodiscus sinensis) and differ from that encoded by kininogen-2 of the Tibetan ground tit (Pseudopodoces humilis) at just a single site (F/L at Position 8). These data would imply that the novel BRP is an amphibian defensive agent against predation by sympatric turtles and also that the primary structure of the avian BK, ornithokinin (RPPGFTPLR), is not invariant within this taxon. Synthetic RVAL-(L1, T6, L8)-bradykinin was found to be an antagonist of BK-induced rat tail artery smooth muscle relaxation acting via the B2-receptor.

Fathers and Parenting

The aim of this chapter is to present a contextual and empirical account of men as fathers in Ireland along with an analysis of recent, relevant developments in policies and laws. Much of parenting of a child occurs in the home. Consequently, how couples inhabit the home greatly influences how their children experience childhood and, in many ways, the story of fatherhood in Ireland is the product of the on-going changes in the domestic sphere. Whether men are driving these changes, embracing or resisting them will provide the substance of much of this chapter. Section one presents basic demographic figures and trends based on census data to answer the questions: who are the fathers, what type of families do the live in and how involved are they in childcare? Section two presents recent research on contemporary fatherhood, both international and, where possible, national. Section three explores the policies and legal measures that affect fathers, their duties and their rights in the home and at work. Section four will discuss the historical legacy of fatherhood that is particular to Ireland. The final section will draw together these threads and ask what might be the future of fatherhood in Ireland, its challenges and possible successes.