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.

Hydrogel-Forming Microneedles Increase in Volume During Swelling in Skin, but Skin Barrier Function Recovery is Unaffected

We describe, for the first time, quantification of in-skin swelling and fluid uptake by hydrogel-forming microneedle (MN) arrays and skin barrier recovery in human volunteers. Such MN arrays, prepared from aqueous blends of hydrolyzed poly(methylvinylether/maleic anhydride) (15%, w/w) and the cross-linker poly(ethyleneglycol) 10,000 Da (7.5%, w/w), were inserted into the skin of human volunteers (n = 15) to depths of approximately 300 μm by gentle hand pressure. The MN arrays swelled in skin, taking up skin interstitial fluid, such that their mass had increased by approximately 30% after 6 h in skin. Importantly, however, skin barrier function recovered within 24 h after MN removal, regardless of how long the MN had been in skin or how much their volume had increased with swelling. Further research on closure of MN-induced micropores is required because transepidermal water loss measurements suggested micropore closure, whereas optical coherence tomography indicated that MN-induced micropores had not closed over, even 24 h after MN had been removed. There were no complaints of skin reactions, adverse events, or strong views against MN use by any of the volunteers. Only some minor erythema was noted after patch removal, although this always resolved within 48 h, and no adverse events were present on follow-up.

Hydrogel-Forming Microneedle Arrays Can Be Effectively Inserted in Skin by Self-Application: A Pilot Study Centred on Pharmacist Intervention and a Patient Information Leaflet

Purpose: To investigate, for the first time, the influence of pharmacist intervention and the use of a patient information leaflet on self-application of hydrogel-forming microneedle arrays by human volunteers without the aid of an applicator device.
Methods: A patient information leaflet was drafted and pharmacist counselling strategy devised. Twenty human volunteers applied 11 × 11 arrays of 400 μm hydrogel-forming microneedle arrays to their own skin following the instructions provided. Skin barrier function disruption was assessed using transepidermal water loss measurements and optical coherence tomography and results compared to those obtained when more experienced researchers applied the microneedles to the volunteers or themselves.
Results: Volunteer self-application of the 400 μm microneedle design resulted in an approximately 30% increase in skin transepidermal water loss, which was not significantly different from that seen with self-application by the more experienced researchers or application to the volunteers. Use of optical coherence tomography showed that self-application of microneedles of the same density (400 μm, 600 μm and 900 μm) led to percentage penetration depths of approximately 75%, 70% and 60%, respectively, though the diameter of the micropores created remained quite constant at approximately 200 μm. Transepidermal water loss progressively increased with increasing height of the applied microneedles and this data, like that for penetration depth, was consistent, regardless of applicant.
Conclusion: We have shown that hydrogel-forming microneedle arrays can be successfully and reproducibly applied by human volunteers given appropriate instruction. If these outcomes were able to be extrapolated to the general patient population, then use of bespoke MN applicator devices may not be necessary, thus possibly enhancing patient compliance.

Honey bee (Apis mellifera) venom induces AIM2 inflammasome activation in human keratinocytes

Following allergen exposure, cytokines and other pro-inflammatory signals play an important role in the immunological cascade leading to allergic sensitization. Inflammasomes sense exogenous and endogenous danger signals and trigger IL-1β and IL-18 activation which in turn shape Th2 responses. Honey bee venom (BV) allergies are very common; however, the local inflammatory cascade leading to the initiation of allergic sensitization is poorly understood. In this study, the local inflammatory cascades in skin after exposure to BV were investigated.

Mechanisms of DNA Damage Response to Targeted Irradiation in Organotypic 3D Skin Cultures

DNA damage (caused by direct cellular exposure and bystander signaling) and the complex pathways involved in its repair are critical events underpinning cellular and tissue response following radiation exposures. There are limited data addressing the dynamics of DNA damage induction and repair in the skin particularly in areas not directly exposed. Here we investigate the mechanisms regulating DNA damage, repair, intracellular signalling and their impact on premature differentiation and development of inflammatory-like response in the irradiated and surrounding areas of a 3D organotypic skin model. Following localized low-LET irradiation (225 kVp X-rays), low levels of 53BP1 foci were observed in the 3D model (3.8±0.28 foci/Gy/cell) with foci persisting and increasing in size up to 48 h post irradiation. In contrast, in cell monolayers 14.2±0.6 foci/Gy/cell and biphasic repair kinetics with repair completed before 24 h was observed. These differences are linked to differences in cellular status with variable level of p21 driving apoptotic signalling in 2D and accelerated differentiation in both the directly irradiated and bystander areas of the 3D model. The signalling pathways utilized by irradiated keratinocytes to induce DNA damage in non-exposed areas of the skin involved the NF-κB transcription factor and its downstream target COX-2.

Identification and Functional Analysis of a Novel Tryptophyllin Peptide from the Skin of the Red-eye Leaf Frog, Agalychnis callidryas

Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation.

Exposure to anthrax toxin alters human leucocyte expression of anthrax toxin receptor 1

Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-γ responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection.

Phylloseptin-PBa—A Novel Broad-Spectrum Antimicrobial Peptide from the Skin Secretion of the Peruvian Purple-Sided Leaf Frog (Phyllomedusa Baltea) Which Exhibits Cancer Cell Cytotoxicity

Antimicrobial peptides from amphibian skin secretion display remarkable broad-spectrum antimicrobial activity and are thus promising for the discovery of new antibiotics. In this study, we report a novel peptide belonging to the phylloseptin family of antimicrobial peptides, from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea, which was named Phylloseptin-PBa. Degenerate primers complementary to putative signal peptide sites of frog skin peptide precursor-encoding cDNAs were designed to interrogate a skin secretion-derived cDNA library from this frog. Subsequently, the peptide was isolated and identified using reverse phase HPLC and MS/MS fragmentation. The synthetic replicate was demonstrated to have activity against S. aureus, E. coli and C. albicans at concentrations of 8, 128 and 8 mg/L, respectively. In addition, it exhibited anti-proliferative activity against the human cancer cell lines, H460, PC3 and U251MG, but was less active against a normal human cell line (HMEC). Furthermore, a haemolysis assay was performed to assess mammalian cell cytotoxicity of Phylloseptin-PBa. This peptide contained a large proportion of α-helical domain, which may explain its antimicrobial and anticancer activities.

Co-expression of NPY and VIP in human dental pulp

Introduction: The regulation of pulpal haemodynamics in health and disease involves sympathetic and parasympathetic mechanisms in which both neuropeptide Y (NPY; a sympathetic vasoconstrictor) and vasoactive intestinal polypeptide (VIP; a parasympathetic vasodilator) may play potential pathophysiological roles. We have previously investigated the levels of NPY or VIP present in human dental pulp tissue and shown that their expression is up-regulated in caries induced pulpal inflammation. Objectives: The aim of this study was to investigate the potential correlation between NPY and VIP levels measured in the same dental pulp samples using radioimmunoassay (RIA). Methods: Pulp tissue was obtained from extracted teeth, classified as follows; healthy (n=22), moderately carious (n=20) and grossly carious (n=26). Samples were processed for RIA by boiling in acetic acid as previously described. The levels of NPY and VIP, measured by RIA, were expressed as ng/gram of pulp tissue. The nature of the relationship between NPY and VIP levels in human pulp tissue was tested by calculating Pearson's product moment correlation coefficient using the linear regression test. Results: Calculation of Pearson product moment correlation coefficient showed a significant negative correlation between NPY and VIP levels in pulp tissue samples from non-carious teeth (p = 0.02, r = -.48). This negative correlation in non-carious teeth changed to a significant positive correlation in carious teeth when the levels of NPY and VIP were compared (p = 0.03, r= 0.311). Conclusions: In non-carious teeth, the negative correlation between NPY and VIP levels is in keeping with the previously described modulatory influence of cholinergic nerves on sympathetic function which may be perturbed as caries develops.

Two Novel Dermaseptin-Like Antimicrobial Peptides with Anticancer Activities from the Skin Secretion of Pachymedusa dacnicolor

The dermaseptin antimicrobial peptide family contains members of 27–34 amino acids in length that have been predominantly isolated from the skins/skin secretions of phyllomedusine leaf frogs. By use of a degenerate primer in Rapid amplification of cDNA ends (RACE) PCR designed to a common conserved domain within the 5′-untranslated regions of previously-characterized dermaseptin encoding cDNAs, two novel members of this peptide family, named dermaseptin-PD-1 and dermaseptin-PD-2, were identified in the skin secretion of the phyllomedusine frog, Pachymedusa dacnicolor. The primary structures of both peptides were predicted from cloned cDNAs, as well as being confirmed by mass spectral analysis of crude skin secretion fractions resulted from reversed-phase high-performance liquid chromatography. Chemically-synthesized replicates of dermaseptin-PD-1 and dermaseptin-PD-2 were investigated for antimicrobial activity using standard model microorganisms (Gram-positive bacteria, Gram-negative bacteria and a yeast) and for cytotoxicity using mammalian red blood cells. The possibility of synergistic effects between the two peptides and their anti-cancer cell proliferation activities were assessed. The peptides exhibited moderate to high inhibition against the growth of the tested microorganisms and cancer cell lines with low haemolytic activity. Synergistic interaction between the two peptides in inhibiting the proliferation of Escherichia coli and human neuronal glioblastoma cell line, U251MG was also manifested.