Cyclotides are disulfide-rich mini-proteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. They typically comprise 28-37 amino acids and are produced from linear precursors, and translational modification via oxidative folding, proteolytic processing and N-C cyclization. Because these plant-derived peptides are resistant to degradation and do exhibit a diverse range of biological activities, they have become important agronomic and industrial objectives. Due to its tolerance to sequence variation, the cyclotide backbone is also potentially useful as a molecular scaffold for protein-engineering applications. Several production options are available for bioactive plant metabolites including natural harvesting, total chemical synthesis, and expression of plant pathways in microbial systems. For the cyclotides with low yields in nature, chemical complexity and lack of knowledge of the complete biosynthetic pathway, however, many of these options are precluded. Plant cell-culture technology shows promise towards the goal of producing therapeutically active cyclotides in quality and quantities required for drug development as they are amenable to process optimization, scale-up, and metabolic engineering. It is conceivable that plant-based production systems may ultimately prove to be the preferred route for the production of native or designed cyclotides, and will contribute towards the development of target-specific drugs.

Cyclotides are naturally occurring mini-proteins that have a cyclic backbone and a knotted arrangement of three disulfide bonds. They are remarkably stable and have a diverse range of therapeutically useful biological activities, including antimicrobial and anti-HIV activity, although their natural function appears to be plant defence agents. Cyclotides are amenable to chemical synthesis; however currently most bioactivity studies have involved the use of peptides extracted from plants. Plant cell culture technology shows promise towards the goal of producing therapeutically active cyclotides in qualities and quantities required for drug development.

The naturally-occurring cyclic cystine-knot microprotein trypsin inhibitors MCoTI-I and MCoTI-II have been synthesised using both thia-zip native chemical ligation and a biomimetic strategy featuring chemoenzymatic cyclisation by an immobilised protease. Engineered analogues have been produced containing a range of substitutions at the P(1) position that redirect specificity towards alternative protease targets whilst retaining excellent to moderate affinity. Furthermore, we report an MCoTI analogue that is a selective low-microM inhibitor of foot-and-mouth-disease virus (FMDV) 3C protease, the first reported peptide-based inhibitor of this important viral enzyme.

ABSTRACT: BACKGROUND: Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a commensal of horses and an opportunistic pathogen in many animals and humans. Some strains produce copious amounts of hyaluronic acid, making S. zooepidemicus an important industrial microorganism for the production of this valuable biopolymer used in the pharmaceutical and cosmetic industry. Encapsulation by hyaluronic acid is considered an important virulence factor in other streptococci, though the importance in S. zooepidemicus remains poorly understood. Proteomics may provide a better understanding of virulence factors in S. zooepidemicus, facilitate the design of better diagnostics and treatments, and guide engineering of superior production strains. RESULTS: Using hyaluronidase to remove the capsule and by optimising cellular lysis, a reference map for S. zooepidemicus was completed. This protocol significantly increased protein recovery, allowing for visualisation of 682 spots and the identification of 86 proteins using mass spectrometry (LC-ESI-MS/MS and MALDI-TOF/TOF); of which 16 were membrane proteins. CONCLUSIONS: The data presented constitute the first reference map for S. zooepidemicus and provide new information on the identity and characteristics of the more abundantly expressed proteins.

Cyclotides are naturally occurring mini-proteins that have a diverse range of therapeutically useful biological activities. Although a choice of approaches is available for cyclotides synthesis; most studies have involved the use of peptides extracted from plants. In order to facilitate the screening for structure-activity studies or to exploit them in drug development, a convenient and reliable route for the biosynthesis of cyclotides is of vital importance. Callus, suspension cultures and hydroponic plants of Oldenlandia affinis were established and have been evaluated for effective cyclotides production processes. The specific accumulation of kalata B1 was affected by cell differentiation as well as agitation; highest accumulation of 2.7mgg(-1) dry weight was detected in agitated hydroponic plant cultures resulting in a productivity of 1.4mgkalataB1l(-1)day(-1).

In this paper, a new technique for online signature verification or identification is proposed. The technique integrates a longest common subsequences (LCSS) detection algorithm which measures the similarity of signature time series into a kernel function for support vector machines (SVM). LCSS offers the possibility to consider the local variability of signals such as the time series of pen-tip coordinates on a graphic tablet, forces on a pen, or inclination angles of a pen measured during a signing process. Consequently, the similarity of two signature time series can be determined in a more reliable way than with other measures. A proprietary database with signatures of 153 test persons and the SVC 2004 benchmark database are used to show the properties of the new SVM-LCSS. We investigate its parameterization and compare it to SVM with other kernel functions such as dynamic time warping (DTW). Our experiments show that SVM with the LCSS kernel authenticate persons very reliably and with a performance which is significantly better than that of the best comparing technique, SVM with DTW kernel.

Cyclotides are disulfide-rich peptides from plants that are exceptionally stable as a result of their unique cyclic cystine knot structural motif. Their natural role is thought to be as plant defence agents, most notably against insect pests, but they also have potential applications in drug design and agriculture. This article identifies gaps in current knowledge on cyclotides and suggests future directions for research into this fascinating family of ultra-stable mini-proteins.

Abstract Objective: The aim of the study was to evaluate the influence of the mode of delivery on female sexuality 12-18 months after childbirth. Methods: Fifty-five primiparae who delivered vaginally without complication and 44 who underwent elective cesarean section after 37 weeks of gestation were included. Sexual function was assessed by a validated self-reported questionnaire, the Female Sexual Function Index (FSFI), 12 months after birth and compared between groups. Additionally, we have analyzed subjective stress variables recorded after birth between the two groups. Results: Women with cesarean section were older (p = 0.002) and had a higher body mass index (BMI)(p =0.02). The total score of the FSFI was not significantly different between the groups. Patients recall of dyspareunia at 3 months after childbirth was higher in those who underwent vaginal delivery (p < 0.001). Conclusions: We suggest that there is no significant difference in sexual function 12-18 months after childbirth between women who delivered vaginally without episiotomy, heavy perineal laceration, or secondary operative interventions and women who underwent elective cesarean section.

Neural networks are often used to process temporal information, i.e., any kind of information related to time series. In many cases, time series contain short-term and long-term trends or behavior. This paper presents a new approach to capture temporal information with various reference periods simultaneously. A least squares approximation of the time series with orthogonal polynomials will be used to describe short-term trends contained in a signal (average, increase, curvature, etc.). Long-term behavior will be modeled with the tapped delay lines of a time-delay neural network (TDNN). This network takes the coefficients of the orthogonal expansion of the approximating polynomial as inputs such considering short-term and long-term information efficiently. The advantages of the method will be demonstrated by means of artificial data and two real-world application examples, the prediction of the user number in a computer network and online tool wear classification in turning.

Observers were scanned while they watched a video of an actor using an object. Three conditions were contrasted in which the same object was used:(i) normally (e.g. using a tennis racket to hit a ball),(ii) in an unusual way (e.g. using a tennis racket to strain spaghetti),(iii) in a pretend play (e.g. playing a tennis racket like a banjo). Observing real and unusual uses of objects activated areas previously seen in studies of tool use including areas associated with a mirror system for action. Observing pretend play activated additional areas previously associated with theory of mind tasks and listening to narrative, including medial prefrontal cortex, posterior superior temporal sulcus and temporal poles. After presentation of each video, observers were asked to name the object as used in the preceding action video (e.g. racket, sieve or banjo). Naming the pretend object elicited activity in medial prefrontal cortex. These results are consistent with proposals that pretend play is a form of communicative narrative, associated with the ability to mentalize. However, this leaves open the question as to whether pretence or mentalizing is the more basic process.

Cyclotides are disulfide-rich mini-proteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. They typically comprise 28-37 amino acids and are produced from linear precursors, and translational modification via oxidative folding, proteolytic processing and N-C cyclization. Because these plant-derived peptides are resistant to degradation and do exhibit a diverse range of biological activities, they have become important agronomic and industrial objectives. Due to its tolerance to sequence variation, the cyclotide backbone is also potentially useful as a molecular scaffold for protein-engineering applications. Several production options are available for bioactive plant metabolites including natural harvesting, total chemical synthesis, and expression of plant pathways in microbial systems. For the cyclotides with low yields in nature, chemical complexity and lack of knowledge of the complete biosynthetic pathway, however, many of these options are precluded. Plant cell-culture technology shows promise towards the goal of producing therapeutically active cyclotides in quality and quantities required for drug development as they are amenable to process optimization, scale-up, and metabolic engineering. It is conceivable that plant-based production systems may ultimately prove to be the preferred route for the production of native or designed cyclotides, and will contribute towards the development of target-specific drugs.

Insulin-like peptide 5 (INSL5) is a two-chain peptide hormone related to insulin and relaxin. It was recently discovered through searches of expressed sequence tag databases and although the full biological significance of INSL5 is still being elucidated, high expression in peripheral tissues such as the colon as well as in the brain and hypothalamus, suggests roles in gut contractility and neuroendocrine signaling. INSL5 activates the relaxin family peptide receptor 4 with high potency, and appears to be the endogenous ligand for this receptor, based on overlapping expression profiles and their apparent co-evolution. Here we have used solution state NMR to characterize the three-dimensional structure of synthetic human INSL5. The structure reveals an insulin/relaxin-like fold with three helical segments that are braced by three disulfide bonds and enclose a hydrophobic core. Furthermore, we characterized in detail the hydrogen bond network and electrostatic interactions between charged groups in INSL5 by NMR-monitored temperature and pH titrations and undertook a comprehensive structural comparison with other members of the relaxin family, thus identifying the conserved structural features of the relaxin fold. The B-chain helix, which is the primary receptor-binding site of the relaxins, is longer in INSL5 than in its close relative relaxin-3. As this feature results in a different positioning of the receptor activation domain ArgB23 and TrpB24 it may be an important contributor to the difference in biological activity observed for these two peptides. Overall the structural studies provide mechanistic insights into the receptor selectivity of this important family of hormones.

Black and white are opposites as are oxidation and reduction. Performing an oxidation, for example, of a sec-alcohol and a reduction of the corresponding ketone in the same vessel without separation of the reagents seems to be an impossible task. Here we show that oxidative cofactor recycling of NADP (+) and reductive regeneration of NADH can be performed simultaneously in the same compartment without significant interference. Regeneration cycles can be run in opposing directions beside each other enabling one-pot transformation of racemic alcohols to one enantiomer via concurrent enantioselective oxidation and asymmetric reduction employing defined alcohol dehydrogenases with opposite stereo- and cofactor-preference. Thus, by careful selection of appropriate enzymes, NADH recycling can be performed in the presence of NADP (+) recycling to achieve overall, for example, deracemisation of sec-alcohols or stereoinversion representing a possible concept for a "green" equivalent to the chemical-intensive Mitsunobu inversion.

As eukaryotes, plants include in innate defense antimicrobial peptides (AMP), usually small cysteine or glycine-rich peptides effective against a wide range of pathogens. The main classes of AMPs are represented by alpha/beta-defensins, lipid-transfer proteins, thionins, cyclotides, snakins and hevein-like, according to amino acid sequence homology. In spite of increasing number of described AMPs from plants, last decade advances in methodologies for gene expression and the huge amounts of genomic, proteomic and other "-omics" data lead to new prospection strategies of novel potential candidates. Organised user-friendly databases are available to be searched and enlarged with newly discovered plant-derived AMPs. Bioinformatics has allowed the application of in silico-associated molecular tools aiming to screen and identify genes coding for these peptides, starting from genome, transcriptomes, proteome or metabolome from various cultivated or wild plants. As expected, crop plants have been the main target for AMP research and application, also because the higher availability of molecular data. However, wild plant species biodiversity and results for AMP search have increased the importance of characterization in native plants. Enormous plant diversity in Brazilian ecosystems summed to croplands provides potential targets to identify novel candidates for plant AMP. Despite these opportunities, bioinformatics tools are restricted to species whose "-omics" are available, otherwise only heterology-based analyses are feasible, as it has been the case of most Brazilian plant AMP prospection research groups. Still rare, but promising results indicate that this research field on Brazilian crop/native species presents a growing trend of application in agriculture, medicine and industry.

Cyclotides are disulfide-rich plant proteins that are exceptional in their cyclic structure; their N and C termini are joined by a peptide bond, forming a continuous circular backbone, which is reinforced by three interlocked disulfide bonds. Cyclotides have been found mainly in the coffee (Rubiaceae) and violet (Violaceae) plant families. Within the Violaceae, cyclotides seem to be widely distributed, but the cyclotide complements of the vast majority of Violaceae species have not yet been explored. This study provides insight into cyclotide occurrence, diversity and biosynthesis in the Violaceae, by identifying mature cyclotide proteins, their precursors and enzymes putatively involved in their biosynthesis in the tribe Rinoreeae and the genus Gloeospermum. Twelve cyclotides from two Panamanian species, Gloeospermum pauciflorum Hekking and Gloeospermum blakeanum (Standl.) Hekking (designated Glopa A-E and Globa A-G, respectively) were characterised through cDNA screening and protein isolation. Screening of cDNA for the oxidative folding enzymes protein-disulfide isomerase (PDI) and thioredoxin (TRX) resulted in positive hits in both species. These enzymes have demonstrated roles in oxidative folding of cyclotides in Rubiaceae, and results presented here indicate that Violaceae plants have evolved similar mechanisms of cyclotide biosynthesis. We also describe PDI and TRX sequences from a third cyclotide-expressing Violaceae species, Viola biflora L., which further support this hypothesis.

Cyclotides are remarkably stable proteins from plants that have a range of pharmaceutical and agricultural applications based both on their various bioactivities and their potential for use as stable protein engineering templates. This article discusses literature on pharmaceutically relevant activities of cyclotides, including anti-HIV, antimicrobial and cytotoxic activities, and evaluates their potential therapeutic applications. Their applications as templates for the design of anti-angiogenic agents for the treatment of cancer, and as anti-infective agents, are also described. Toxic effects of cyclotides, whose native function is as insecticidal agents, can be removed by simple mutagenesis, thus rationalizing the apparent conundrum of proposing insecticidal agents as leads for human therapeutics.

Cyclotides are disulfide-rich peptides from plants that are exceptionally stable as a result of their unique cyclic cystine knot structural motif. Their natural role is thought to be as plant defence agents, most notably against insect pests, but they also have potential applications in drug design and agriculture. This article identifies gaps in current knowledge on cyclotides and suggests future directions for research into this fascinating family of ultra-stable mini-proteins.

The cyclotides are a large family of circular mini-proteins containing a cystine knot motif. They are expressed in plants as defence-related proteins, with insecticidal activity. Here we investigate their role in membrane interaction and disruption. Kalata B1, a prototypic cyclotide, was found to induce leakage of the self-quenching fluorophore, carboxyfluorescein, from phospholipid vesicles. Alanine-scanning mutagenesis of kalata B1 showed that residues essential for lytic activity are clustered, forming a bioactive face. Kalata B1 was sequestered at the membrane surface and showed slow dissociation from vesicles. Electrophysiological experiments showed that conductive pores were induced in liposome patches on incubation with kalata B1. The conductance calculated from the current-voltage relationship indicated that the diameter of the pores formed in the bilayer patches is 41-47 A. Collectively, the findings provide a mechanistic explanation for the diversity of biological functions ascribed to this fascinating family of ultrastable macrocyclic peptides.

Cyclotides are cyclic disulfide rich mini-proteins found in various Rubiaceae (coffee family), Violaceae (violet family) and Cucurbitaceae (squash family) plant species. Within the Violaceae, cyclotides have been found in numerous species of the genus Viola as well as species from two other genera, namely Hybanthus and Leonia. This is the first in-depth report of cyclotides in the genus Melicytus (Violaceae). We present the chromatographic profiles of extracts of eight Melicytus species and one Melicytus hybrid that were found to contain these circular peptides. We isolated and characterised five novel cyclotides (mra1 to mra5) from the aerial parts of a common New Zealand tree, Melicytus ramiflorus. All five peptides show the characteristics of the bracelet subfamily of cyclotides. Furthermore, we isolated 17 non-redundant cDNA clones from the leaves of Melicytus ramiflorus encoding cyclotide prepropeptides. This detailed report on the presence of cyclotides in several species of the genus Melicytus further strengthens our hypothesis that cyclotides are ubiquitous in Violaceae family plants and provides additional insight into the biochemical processing mechanisms that produce the cyclic protein backbone of this unique family of ultra-stable plant proteins.

Cyclotides are disulfide-rich mini-proteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. They typically comprise 28-37 amino acids and are produced from linear precursors, and translational modification via oxidative folding, proteolytic processing and N-C cyclization. Because these plant-derived peptides are resistant to degradation and do exhibit a diverse range of biological activities, they have become important agronomic and industrial objectives. Due to its tolerance to sequence variation, the cyclotide backbone is also potentially useful as a molecular scaffold for protein-engineering applications. Several production options are available for bioactive plant metabolites including natural harvesting, total chemical synthesis, and expression of plant pathways in microbial systems. For the cyclotides with low yields in nature, chemical complexity and lack of knowledge of the complete biosynthetic pathway, however, many of these options are precluded. Plant cell-culture technology shows promise towards the goal of producing therapeutically active cyclotides in quality and quantities required for drug development as they are amenable to process optimization, scale-up, and metabolic engineering. It is conceivable that plant-based production systems may ultimately prove to be the preferred route for the production of native or designed cyclotides, and will contribute towards the development of target-specific drugs.

The cyclic cystine knot motif, as defined by the cyclotide peptide family, is an attractive scaffold for protein engineering. To date, however, the utilisation of this scaffold has been limited by the inability to synthesise members of the most diverse and biologically active subfamily, the bracelet cyclotides. This study describes the synthesis and first direct oxidative folding of a bracelet cyclotide-cycloviolacin O2-and thus provides an efficient method for exploring the most potent cyclic cystine knot peptides. The linear chain of cycloviolacin O2 was assembled by solid-phase Fmoc peptide synthesis and cyclised by thioester-mediated native chemical ligation, and the inherent difficulties of folding bracelet cyclotides were successfully overcome in a single-step reaction. The folding pathway was characterised and was found to include predominating fully oxidised intermediates that slowly converted to the native peptide structure.