Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs

Brevinins are peptides of 24 amino acid residues, originally isolated from the skin of the Oriental frog, Rana brevipoda porsa, by nature of their microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi. cDNA libraries were constructed from lyophilized skin secretion of three, unstudied species of Chinese frog, Odorrana schmackeri, Odorrana versabilis and Pelophylax plancyi fukienensis, using our recently developed technique. In this report, we describe the “shotgun” cloning of novel brevinins by means of 3'-RACE, using a “universal” degenerate primer directed towards a highly conserved nucleic acid sequence domain within the 5'-untranslated region of previously characterized frog skin peptide cDNAs. Novel brevinins, deduced from cloned cDNA open-reading frames, were subsequently identified as mature peptides in the same samples of respective species skin secretions. Bioinformatic analysis of both prepro-brevinin nucleic acid sequences and translated open-reading frame amino acid sequences revealed a highly conserved signal peptide domain and a hypervariable anti-microbial peptide-encoding domain. The experimental approach described here can thus rapidly provide robust structural data on skin anti-microbial peptides without harming the donor amphibians.

Novel frenatins from the skin of the Australasian Giant White-lipped tree frog, Litoria infrafrenata: cloning of precursor cDNAs and identification in defensive skin secretion.

The Australasian anuran amphibian genus Litoria, contains many phenotypically-diverse species as a result of radial evolution of an ancestral species into different biotopes much in the manner of the indigenous marsupial mammals. In common with members of the Central/South American genus Phyllomedusa, their specialized skin granular glands are factories for the production of a plethora of biologically-active peptides. Here we report a more detailed study of those present in the defensive skin secretion of the Australasian giant white-lipped tree frog, Litoria infrafrenata, and, for the first time, we have identified three novel frenatins by deduction of primary structures from cDNAs that were cloned from a library constructed from lyophilized skin secretion using a recently-developed technique. All open-reading frames consisted of a putative signal peptide and an acidic pro-region followed by a single copy of a frenatin peptide. Processed peptides corresponding in molecular mass to the deduced molecular masses of frenatins (named 1.1, 3, 3.1 and 4.1) were identified in the same secretion sample using HPLC and mass spectroscopy. The application of this technique thus permits parallel peptidomic and transcriptomic analyzes on the same lyophilized skin secretion sample circumventing sacrifice of specimens from endangered herpetofauna.

Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.

Rapid identification of precursor cDNAs encoding five structural classes of antimicrobial peptides from pickerel frog (Rana palustris) skin secretion by single step “shotgun” cloning.

The skin secretion of the North American pickerel frog (Rana palustris) has long been known to have pronounced noxious/toxic properties and to be highly effective in defence against predators and against other sympatric amphibians. As it consists largely of a complex mixture of peptides, it has been subjected to systematic peptidomic study but there has been little focus on molecular cloning of peptide-encoding cDNAs and by deduction, the biosynthetic precursors that they encode. Here, we demonstrate that the cDNAs encoding the five major structural families of antimicrobial peptides can be elucidated by a single step “shotgun” cloning approach using a cDNA library constructed from the source material of the peptidomic studies—the defensive skin secretion itself. Using a degenerate primer pool designed to a highly conserved nucleic acid sequence 5' to the initiation codon of known antimicrobial peptide precursor transcripts, we amplified cDNA sequences representing five major classes of antimicrobial peptides, such as esculentins, brevinins, ranatuerins, palustrins and temporins. Bioinformatic comparisons of precursor open-reading frames and nucleic acid sequences revealed high degrees of structural similarities between analogous peptides of R. palustris and the Chinese bamboo odorous frog, Rana versabilis. This approach thus constitutes a robust technique that can be used either alone or ideally, in parallel with peptidomic analysis of skin secretion, to rapidly extract primary structural information on amphibian skin secretion peptides and their biosynthetic precursors.

Cloning of maximakinin precursor cDNAs from Chinese toad, Bombina maxima, venom.

Using a novel technique that we have developed for cloning of amphibian skin secretion peptide cDNAs from lyophilized samples, we report here that maximakinin (DLPKINRKGP-bradykinin) is encoded by two different cDNAs, named BMK-1 and BMK-2, containing either four tandem repeat sequences or a single copy. The open reading frames of both precursor cDNAs were found to be 152 and 116 amino acid residues, respectively. These data provide evidence that the structural diversity of peptides in amphibian skin secretions arising from molecular evolutionary events, can be mediated by parallel diversity in encoding mRNAs that in itself may reflect serial gene duplications.

The structural organization of aurein precursor cDNAs from the skin secretion of the Australian green and golden bell frog, Litoria aurea.

Aureins are a family of peptides (13-25 residues), some of which possess potent antimicrobial and anti-cancer properties, which have been classified into 5 subgroups based upon primary structural similarities. They were originally isolated from the defensive skin secretions of the closely related Australian bell frogs, Litoria aurea and Litoria raniformis, and of the 23 aurein peptides identified, 10 are common to both species. Using a recently developed technique, we have constructed a cDNA library from the defensive secretion of the green and golden bell frog, L. aurea, and successfully cloned a range of aurein precursor transcripts containing entire open-reading frames. All open-reading frames consisted of a putative signal peptide and an acidic pro-region followed by a single copy of aurein. The deduced precursor structures for the most active aureins (2.2 and 3.1) confirmed the presence of a C-terminal amidation motif whereas that of aurein 5.3 did not. Processed peptides corresponding in molecular mass to aureins 2.2, 2.3, 2.5, 3.1 and 5.3 were identified in the same secretion sample using LC/MS. The application of this technique thus permits parallel peptidomic and transcriptomic analyses on the same lyophilized skin secretion sample circumventing sacrifice of specimens of endangered herpetofauna.

Head-SAR dependence on spectacle frame shape for operator of 450 MHz personal radio

Power deposition in the head of a user wearing metal-framed spectacles was calculated with a 450 MHz personal radio transmitting in close proximity. Peak tissue SAR in the head depended on lens shape whether circular half-rim or rectangular with 70 and 174% increases, respectively, compared to the spectacle-free case. However, localised screening occurred with square frames, with a 40% reduction of peak SAR in the eye closest to the antenna.

Modeling energy consumption in error-prone IEEE 802.11-based wireless ad-hoc networks

In the IEEE 802.11 MAC layer protocol, there are different trade-off points between the number of nodes competing for the medium and the network capacity provided to them. There is also a trade-off between the wireless channel condition during the transmission period and the energy consumption of the nodes. Current approaches at modeling energy consumption in 802.11 based networks do not consider the influence of the channel condition on all types of frames (control and data) in the WLAN. Nor do they consider the effect on the different MAC and PHY schemes that can occur in 802.11 networks. In this paper, we investigate energy consumption corresponding to the number of competing nodes in IEEE 802.11's MAC and PHY layers in error-prone wireless channel conditions, and present a new energy consumption model. Analysis of the power consumed by each type of MAC and PHY over different bit error rates shows that the parameters in these layers play a critical role in determining the overall energy consumption of the ad-hoc network. The goal of this research is not only to compare the energy consumption using exact formulae in saturated IEEE 802.11-based DCF networks under varying numbers of competing nodes, but also, as the results show, to demonstrate that channel errors have a significant impact on the energy consumption.

The Open Method of Coordination on Health Care After the Lisbon Strategy II: Towards a Neoliberal Framing?

This paper undertakes a content analysis of the discourse on the Open Method of Coordination on Health Care (OMC/HC) in order to show how equity and solidarity are increasingly linked to optimisation and, as such, how neoliberalism increasingly frames health care. Some of the side-effects of this reframing for politics are highlighted: legitimating and extending EU governance, reducing the space for oppositional formations and limited citizenship. The analysis begins by interrogating the broader context of the Lisbon Strategy II, after which the techniques of the OMC/HC and its substantive outputs are analysed.

Object tracking using SIFT features and mean shift

A scale invariant feature transform (SIFT) based mean shift algorithm is presented for object tracking in real scenarios. SIFT features are used to correspond the region of interests across frames. Meanwhile, mean shift is applied to conduct similarity search via color histograms. The probability distributions from these two measurements are evaluated in an expectation–maximization scheme so as to achieve maximum likelihood estimation of similar regions. This mutual support mechanism can lead to consistent tracking performance if one of the two measurements becomes unstable. Experimental work demonstrates that the proposed mean shift/SIFT strategy improves the tracking performance of the classical mean shift and SIFT tracking algorithms in complicated real scenarios.

A programmable architecture for layered multimedia streams in IPv6 networks

A new configurable architecture is presented that offers multiple levels of video playback by accommodating variable levels of network utilization and bandwidth. By utilizing scalable MPEG-4 encoding at the network edge and using specific video delivery protocols, media streaming components are merged to fully optimize video playback for IPv6 networks, thus improving QoS. This is achieved by introducing “programmable network functionality” (PNF) which splits layered video transmission and distributes it evenly over available bandwidth, reducing packet loss and delay caused by out-of-profile DiffServ classes. An FPGA design is given which gives improved performance, e.g. link utilization, end-to-end delay, and that during congestion, improves on-time delivery of video frames by up to 80% when compared to current “static” DiffServ.

Hierarchical Video Summarization in Reference Subspace

In this paper, a hierarchical video structure summarization approach using Laplacian Eigenmap is proposed, where a small set of reference frames is selected from the video sequence to form a reference subspace to measure the dissimilarity between two arbitrary frames. In the proposed summarization scheme, the shot-level key frames are first detected from the continuity of inter-frame dissimilarity, and the sub-shot level and scene level representative frames are then summarized by using K-mean clustering. The experiment is carried on both test videos and movies, and the results show that in comparison with a similar approach using latent semantic analysis, the proposed approach using Laplacian Eigenmap can achieve a better recall rate in keyframe detection, and gives an efficient hierarchical summarization at sub shot, shot and scene levels subsequently.

Hylambatin and (Thr)11-hylambatin from the skin secretion of the African hyperoliid frog, Kassina maculata: Structural characterisation, precursor cDNA cloning and preliminary pharmacological testing

The tachykinins hylambatin and (Thr)11-hylambatin have been isolated from the defensive skin secretion of the African hyperoliid frog, Kassina maculata,. Hylambatin (DPPDPNRFYGMMamide) is revised in structure from the original sequence by a single site substitution (Asn/Asp at position 6), and (Thr)11-hylambatin, a novel tachykinin, differs in structure from hylambatin by a single Thr/Met substitution. (Thr)11-hylambatin is five- to ten-fold more abundant than hylambatin in secretions. Synthetic replicates of both peptides were active in smooth muscle preparations including the rat tail artery, rat ileum and bovine trachea. While hylambatin displayed activity consistent with an NK1-receptor ligand, (Thr)11-hylambatin was more active than either substance P or neurokinin A in both NK1- and NK-2 receptor rich preparations. Incorporation of a threoninyl residue rather than the canonical leucyl residue at the penultimate position in both substance P and neurokinin A, generated active ligands in both arterial and intestinal smooth muscle preparations. Hylambatin precursor cDNAs, designated HYBN-1 and HYBN-2, respectively, were cloned from a skin library by 3'- and 5'-RACE reactions. Both were highly-homologous containing open-reading frames of 66 amino acids encoding single copies of either hylambatin or (Thr)11-hylambatin. These data reveal a hitherto unrecognized structure/activity attribute of mammalian tachykinin receptors revealed though discovery of a novel amphibian skin-derived, site-substituted peptide ligand.

Spatial Perception and Cognition in Multichannel Audio for Electroacoustic Music

Listeners experience electroacoustic music as full of significance and meaning, and they experience spatiality as one of the factors contributing to its meaningfulness. If we want to understand spatiality in electroacoustic music, we must understand how the listener’s mental processes give rise to the experience of meaning. In electroacoustic music as in everyday life, these mental processes unite the peripheral auditory system with human spatial cognition. In the discussion that follows we consider a range of the listener’s mental processes relating space and meaning from the perceptual attributes of spatial imagery to the spatial reference frames for places and navigation. When considering multichannel loudspeaker systems in particular, an important part of the discussion is focused on the distinctive and idiomatic ways in which this particular mode of sound production contributes to and situates meaning. These idiosyncrasies include the phenomenon of image dispersion, the important consequences of the precedence effect and the influence of source characteristics on spatial imagery. These are discussed in close relation to the practicalities of artistic practice and to the potential for artistic meaning experienced by the listener.

On-line Emotion Recognition in a 3-D Activation-Valence-Time Continuum using Acoustic and Linguistic Cues

For many applications of emotion recognition, such as virtual agents, the system must select responses while the user is speaking. This requires reliable on-line recognition of the user’s affect. However most emotion recognition systems are based on turnwise processing. We present a novel approach to on-line emotion recognition from speech using Long Short-Term Memory Recurrent Neural Networks. Emotion is recognised frame-wise in a two-dimensional valence-activation continuum. In contrast to current state-of-the-art approaches, recognition is performed on low-level signal frames, similar to those used for speech recognition. No statistical functionals are applied to low-level feature contours. Framing at a higher level is therefore unnecessary and regression outputs can be produced in real-time for every low-level input frame. We also investigate the benefits of including linguistic features on the signal frame level obtained by a keyword spotter.