Enhancement Factor for Gas Absorption in a Finite Liquid Layer. Part 2: First- and Second-Order Reactions in a Liquid in Plug Flow

Gas absorption accompanied by an irreversible chemical reaction of first-order or second-order in a liquid layer of finite thickness in plug flow has been investigated. The analytical solution to the enhancement factor has been derived for the case of a first-order reaction, and the exact solution to the enhancement factor has been obtained via numerical simulation for the case of a second-order reaction. The enhancement factor in both cases is presented as a function of the Fourier number and tends to deviate from the prediction of the existing enhancement factor expressions based on the penetration theory at Fourier numbers above 0.1 due to the absence of a well-mixed bulk region in the liquid layer. Approximate enhancement factor expressions that describe the analytical and exact solutions with an accuracy of 5?% and 9?%, respectively, have been proposed.

Antimicrobial peptides and alytesin are co-secreted from the venom of the Midwife toad, Alytes maurus (Alytidae, Anura): Implications for the evolution of frog skin defensive secretions

The skin secretions of frogs and toads (Anura) have long been a known source of a vast abundance of bioactive substances. In the past decade, transcriptome data of the granular glands of anuran skin has given new impetus to investigations of the putative constituent peptides. Alytes obstetricans was recently investigated and novel peptides with antimicrobial activity were isolated and functionally characterised. However, genetic data for the evolutionarily ancient lineage to which Alytes belongs (midwife toads; Alytidae) remains unavailable.

Here we present the first such genetic data for Alytidae, derived via the granular gland transcriptome of a closely-related species of midwife toad, Alytes maurus. First, we present nucleotide sequences of the entire peptide precursors for four novel antimicrobial peptides (AMPs). The two precursors resemble those from Bombinatoridae in both their structural architecture and amino acid sequence. Each precursor comprises two AMPs as tandem repeats, with a member of the alyteserin-1 family (alyteserin-1Ma: GFKEVLKADLGSLVKGIAAHVAN-NH2 or alyteserin-1Mb: GFKEVLKAGLGSLVKGIPAHVAN-NH2) followed by its corresponding member from the alyteserin-2 family (alyteserin-2Ma: FIGKLISAASGLLSHL-NH2 or alyteserin-2Mb: ILGAIIPLVSGLLSHL-NH2). Synthetic replicates of the four AMPs possessed minimal inhibitory concentrations (MICs) ranging from 9.5 to 300 µM, with the most potent being alyteserin-2Ma. Second, we also cloned the cDNA encoding an alytesin precursor, with the active alytesin exhibiting high sequence identity to bombesin-related peptides from other frogs. All putative mature peptide sequences were confirmed to be present in the skin secretion via LC/MS.

The close structural resemblance of the alyteserin genes that we isolated for A. maurus with those of Bombina provide independent molecular evidence for a close evolutionary relationship between these genera as well as more support for the convergent evolution of the AMP system within anurans. In contrast to the more evolutionarily conserved nature of neuropeptides (including alytesin, which we also isolated), the more variable nature of the AMP system together with the sporadic distribution of AMPs among anuran amphibians fuels in part our hypothesis that the latter system was co-opted secondarily to fulfil a function in the innate immune system, having originally evolved for defence against potential macropredators.

The chains of the heterodimeric amphibian skin antimicrobial peptide, distinctin, are encoded by separate messenger RNAs

Using a primer to a conserved nucleotide sequence of previously-cloned skin peptides of Phyllomedusa species, two distinct cDNAs were “shotgun” cloned from a skin secretion-derived cDNA library of the frog, Phyllomedusa burmeisteri. The two ORFs separately encode chains A and B of an analog of the previously-reported heterodimeric peptide, distinctin. LC-MS/MS analysis of native versus dithiotreitol reduced crude venom, confirmed the predicted primary sequences as well as the cystine link between the two monomers. Distinctin predominantly exists in the venom as a heterodimer (A-B), neither of the constituent peptides were detected as monomer, whereas of the two possible homodimers (A-A or B-B), only B-B was detected in comparatively low quantity. In vitro dimerization of synthetic replicates of the monomers demonstrated that besides heterodimer, both homodimers are also formed in considerable amounts. Distinctin is the first example of an amphibian skin dimeric peptide that is formed by covalent linkage of two chains that are the products of different mRNAs. How this phenomenon occurs in vivo, to exclude significant homodimer formation, is unclear at present but a “favored steric state” type of interaction between chains is most likely.

Determining the role of mononuclear phagocytes in prion neuroinvasion from the skin

Many prion diseases are acquired by peripheral exposure, and skin lesions are an effective route of transmission. Following exposure, early prion replication, upon FDCs in the draining LN is obligatory for the spread of disease to the brain. However, the mechanism by which prions are conveyed to the draining LN is uncertain. Here, transgenic mice were used, in which langerin(+) cells, including epidermal LCs and langerin(+) classical DCs, were specifically depleted. These were used in parallel with transgenic mice, in which nonepidermal CD11c(+) cells were specifically depleted. Our data show that prion pathogenesis, following exposure via skin scarification, occurred independently of LC and other langerin(+) cells. However, the depletion of nonepidermal CD11c(+) cells impaired the early accumulation of prions in the draining LN, implying a role for these cells in the propagation of prions from the skin. Therefore, together, these data suggest that the propagation of prions from the skin to the draining LN occurs via dermal classical DCs, independently of langerin(+) cells.

Skin langerin+ dendritic cells transport intradermally injected anti-DEC-205 antibodies but are not essential for subsequent cytotoxic CD8+ T cell responses

Incorporation of Ags by dendritic cells (DCs) increases when Ags are targeted to endocytic receptors by mAbs. We have previously demonstrated in the mouse that mAbs against C-type lectins administered intradermally are taken up by epidermal Langerhans cells (LCs), dermal Langerin(neg) DCs, and dermal Langerin(+) DCs in situ. However, the relative contribution of these skin DC subsets to the induction of immune responses after Ag targeting has not been addressed in vivo. We show in this study that murine epidermal LCs and dermal DCs transport intradermally injected mAbs against the lectin receptor DEC-205/CD205 in vivo. Skin DCs targeted in situ with mAbs migrated through lymphatic vessels in steady state and inflammation. In the skin-draining lymph nodes, targeting mAbs were found in resident CD8a(+) DCs and in migrating skin DCs. More than 70% of targeted DCs expressed Langerin, including dermal Langerin(+) DCs and LCs. Numbers of targeted skin DCs in the nodes increased 2-3-fold when skin was topically inflamed by the TLR7 agonist imiquimod. Complete removal of the site where OVA-coupled anti-DEC-205 had been injected decreased endogenous cytotoxic responses against OVA peptide-loaded target cells by 40-50%. Surprisingly, selective ablation of all Langerin(+) skin DCs in Langerin-DTR knock-in mice did not affect such responses independently of the adjuvant chosen. Thus, in cutaneous immunization strategies where Ag is targeted to DCs, Langerin(+) skin DCs play a major role in transport of anti-DEC-205 mAb, although Langerin(neg) dermal DCs and CD8a(+) DCs are sufficient to subsequent CD8(+) T cell responses.

The structures of four bombesins and their cloned precursor-encoding cDNAs from acid-solvated skin secretion of the European yellow-bellied toad, Bombina variegata

Four different bombesins (bombesin, His(6)-bombesin, Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin) have been identified by a systematic sequencing study of peptides in reverse phase HPLC fractions of the skin secretion of the European yellow-bellied toad, Bombina variegata, that had been solvated in 0.1% (v/v) aqueous trifluoroacetic acid (TFA) and stored frozen at -20°C for 12 years. By using a 3'- and 5'-RACE PCR strategy, the corresponding biosynthetic precursor-encoding cDNAs of all four peptides were cloned from a cDNA library made from the same long-term frozen, acid-solvated skin secretion sample following thawing and lyophilization. Canonical bombesin and His(6)-bombesin are classical bombesin sub-family members, whereas Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin, belong to the litorin/ranatensin sub-family of bombesin-like peptides (BLPs). Assignment of these peptides to respective sub-families, was based upon both their primary structural similarities and their comparative pharmacological activities. An interesting observation in this study, was that the nucleotide sequences of the open-reading frames of cloned cDNAs encoding bombesin and its His(6)-substituted analog, were identical except for a single base that was responsible for the change observed at the position 6 residue in the mature peptide from Asn to His. In contrast, the precursor cDNA nucleotide sequences encoding the Phe(13)-bombesins, exhibited 53 base differences. The pharmacological activities of synthetic replicates of each bombesin were compared using two different mammalian smooth muscle preparations and all four peptides were found to be active. However, there were significant differences in their relative potencies.

Assessment of cell-mediated immunity in a British population using multiple skin test antigens

An in vivo method of assessing the competence of the cell-mediated immune system (Multitest CMI) was used in 200 healthy volunteers (age range 17-88 years). The profile of reactivity to seven individual antigens was determined. A positive reaction was obtained in 96.5% of the subjects who reacted positively to at least one antigen with 78% reacting to two or more antigens. The number of positive responses and the degree of reactivity was significantly reduced in elderly subjects and in females aged 17-65 years. The Multitest CMI system provides a rapid and convenient method of assessing cell-mediated immunity (CMI) in vivo and could have a wide range of applications in the investigation of immunological, infective and neoplastic conditions.

Efficient differentiation into skin cells of bone marrow cells recovered in a pellet after density gradient fractionation

We had previously demonstrated the participation of whole bone marrow cells from adult mice in the reconstitution of skin, including the epidermis and hair follicles. To get an insight into cell populations that give rise to the epithelial components of the reconstituted skin, we fractionated bone marrow cells derived from green fluorescent protein-transgenic mice by density gradient. Unexpectedly, we found that a substantial amount of mononucleated cells (approximately 30%) was recovered in the pellet fraction and that the cells in the pellet fraction preferentially differentiated into epithelial components of skin, rather than the cells in the mononuclear cell fraction. The pellet fraction contained more CD45-negative (thus uncommitted to the hematopoietic cell lineage) cells than the mononuclear cell fraction. These results indicate that density gradient fractionation results in significant loss of specific progenitor cells into the usually discarded pellet fraction.

Stellar Surface Magneto-Convection as a Source of Astrophysical Noise. I. Multi-component Parameterisation of Absorption Line Profiles

We outline our techniques to characterise photospheric granulation as an astrophysical noise source. A four component parameterisation of granulation is developed that can be used to reconstruct stellar line asymmetries and radial velocity shifts due to photospheric convective motions. The four components are made up of absorption line profiles calculated for granules, magnetic intergranular lanes, non-magnetic intergranular lanes, and magnetic bright points at disc centre. These components are constructed by averaging Fe I $6302 \mathrm{\AA}$ magnetically sensitive absorption line profiles output from detailed radiative transport calculations of the solar photosphere. Each of the four categories adopted are based on magnetic field and continuum intensity limits determined from examining three-dimensional magnetohydrodynamic simulations with an average magnetic flux of $200 \mathrm{G}$. Using these four component line profiles we accurately reconstruct granulation profiles, produced from modelling 12 x 12 Mm$^2$ areas on the solar surface, to within $\sim \pm$ 20 cm s$^{-1}$ on a $\sim$ 100 m s$^{-1}$ granulation signal. We have also successfully reconstructed granulation profiles from a $50 \mathrm{G}$ simulation using the parameterised line profiles from the $200 \mathrm{G}$ average magnetic field simulation. This test demonstrates applicability of the characterisation to a range of magnetic stellar activity levels.

Vasorelaxin: A Novel Arterial Smooth Muscle-Relaxing Eicosapeptide from the Skin Secretion of the Chinese Piebald Odorous Frog (Odorrana schmackeri)

The defensive skin secretions of amphibians are a rich resource for the discovery of novel, bioactive peptides. Here we report the identification of a novel vascular smooth muscle-relaxing peptide, named vasorelaxin, from the skin secretion of the Chinese piebald odorous frog, Odorrana schmackeri. Vasorelaxin consists of 20 amino acid residues, SRVVKCSGFRPGSPDSREFC, with a disulfide-bridge between Cys-6 and Cys-20. The structure of its biosynthetic precursor was deduced from cloned skin cDNA and consists of 67 amino acid residues encoding a single copy of vasorelaxin (vasorelaxin, accession number: HE860494). Synthetic vasorelaxin caused a profound relaxation of rat arterial smooth muscle with an EC50 of 6.76 nM.

Influence of skin model on in vitro performance of drug-loaded soluble microneedle arrays

A plethora of studies have described the in vitro assessment of dissolving microneedle (MN) arrays for enhanced transdermal drug delivery, utilising a wide variety of model membranes as a representation of the skin barrier. However, to date, no discussion has taken place with regard to the choice of model skin membrane and the impact this may have on the evaluation of MN performance. In this study, we have, for the first time, critically assessed the most common types of in vitro skin permeation models - a synthetic hydrophobic membrane (Silescol(®) of 75 µm) and neonatal porcine skin of definable thickness (300-350 µm and 700-750 µm) - for evaluating the performance of drug loaded dissolving poly (methyl vinyl ether co maleic acid) (PMVE/MA) MN arrays. It was found that the choice of in vitro skin model had a significant effect on the permeation of a wide range of small hydrophilic molecules released from dissolving MNs. For example, when Silescol(®) was used as the model membrane, the cumulative percentage permeation of methylene blue 24h after the application of dissolvable MNs was found to be only approximately 3.7% of the total methylene blue loaded into the MN device. In comparison, when dermatomed and full thickness neonatal porcine skin were used as a skin model, approximately 67.4% and 47.5% of methylene blue loaded into the MN device was delivered across the skin 24h after the application of MN arrays, respectively. The application of methylene blue loaded MN arrays in a rat model in vivo revealed that the extent of MN-mediated percutaneous delivery achieved was most similar to that predicted from the in vitro investigations employing dermatomed neonatal porcine skin (300-350 µm) as the model skin membrane. On the basis of these results, a wider discussion within the MN community will be necessary to standardise the experimental protocols used for the evaluation and comparison of MN devices.

Molecular cloning of the trypsin inhibitor from the skin secretion of the Madagascan Tomato Frog, Dyscophus guineti (Microhylidae), and insights into its potential defensive role

In this study, we investigate the skin secretion of the Madagascan Tomato Frog, Dyscophus guineti, which is characterized by its peculiarly adhesive and viscous nature, with a view toward the function of the member of the Kunitz/bovine pancreatic trypsin inhibitor family (BPTI) it is known to contain. Using “shotgun” cloning of a skin secretion-derived cDNA library, we obtained the full-length sequence of the respective precursor that encodes this trypsin inhibitor. Furthermore, we demonstrated that this enzyme has inhibitory activity against trypsin, but not against thrombin, and also has no antimicrobial activity. Moreover, we confirm that it appears to be the only bioactive peptide in the skin secretion of this species. Using these observations, we attempt to posit a role for this inhibitor. In particular, we hypothesize that the trypsin inhibitor in D. guineti (and possibly other microhylid frogs) maintains the soluble state of the skin secretion during storage in the glands. Upon discharge of the secretion, the trypsin inhibitor, which occurs in low concentrations, can no longer prevent the polymerisation process of other yet unidentified skin proteins, thereby resulting in the conversion of the secretion to its final glue-like state. Thus, the major defensive value of the skin secretion appears to be mechanical, impeding ingestion through a combination of adhesion and the body inflation typical for some microhylid frogs rather than chemical through antimicrobial activity or toxicity.