Internet-delivered cognitive behaviour therapy for adolescents with insomnia comorbid to psychiatric conditions: A non-randomised trial.

Insomnia is highly prevalent among adolescents with psychiatric conditions and is known to aggravate psychiatric symptoms. Research on cognitive behaviour therapy for adolescents with comorbid insomnia (CBT-I) is still limited. The aim of this study was to investigate feasibility and preliminary effects of internet-delivered CBT for adolescents with insomnia comorbid to a psychiatric condition. Twenty-one patients (13-17 years) with comorbid insomnia were recruited from Child and Adolescent Psychiatry. All patients received 7 weeks of internet-delivered CBT-I with therapist support. Outcomes were assessed at baseline, post-treatment, and at a 4-month follow-up. The proportion of completed assessments was overall acceptable. Participants on average completed 4.48 (sd = 1.97) of the seven treatment modules and therapists on average spent 12.80 minutes (sd = 6.23) per patient and week. Results showed large statistically significant improvements on insomnia severity, sleep efficiency, sleep onset latency and sleep quality. Medium to large improvements were also seen on the psychiatric symptoms of depression, obsessive-compulsive symptoms, interpersonal sensitivity, paranoid ideation and psychoticism. These findings indicate that internet-delivered CBT is feasible and potentially promising for improving sleep and reducing psychiatric symptoms in adolescent psychiatric patients with insomnia and co-morbid psychiatric disorders. A larger randomised trial is warranted to verify these preliminary results.

Orellanine specifically targets renal clear cell carcinoma.

Renal cell carcinoma (RCC), arising from the proximal tubule in the kidney, accounts for approximately 85% of kidney cancers and causes over 140,000 annual deaths worldwide. In the last decade, several new therapies have been identified for treatment of metastatic RCC. Although these therapies increase survival time compared to standard care, none of them has curative properties. The nephrotoxin orellanine specifically targets proximal tubular epithelial cells, leaving other organs unaffected. We therefore hypothesized that the selective toxicity of orellanine extends to clear cell RCC (ccRCC) cells since they emanate from proximal tubular cells. Orellanine would thus target both primary and metastatic ccRCC in vitro and in vivo. We found that orellanine induces dose-dependent cell death in proximal tubular cells and in all ccRCC cells tested, both primary and cell lines, with no toxicity detected in control cells. The toxic action of orellanine involve decreased protein synthesis, disrupted cell metabolism and induction of apoptosis. In nude rats carrying human ccRCC xenografts, brief orellanine treatment eliminated more than 90% of viable tumor mass compared to control rats. This identifies orellanine as a potential treatment concept for ccRCC patients on dialysis, due to its unique selective toxicity towards ccRCC.

Analysis of the mushroom nephrotoxin orellanine and its glucosides.

Orellanine is a nephrotoxin found in various Cortinaceae mushroom species. Unintentional consumption after these species were confused with edible mushrooms such as Cantharellus tubaeformis has caused several casualties. In this work, a quantitative HPLC-ESI-MS/MS method for total orellanine in Cortinarius rubellus, spiked blood plasma, and a mushroom stew prepared from C. tubaeformis with the addition of a single specimen of C. rubellus is presented. The existence of mono- and diglucosylated orellanine in C. rubellus was also proven, although quantitative analysis could not be obtained for the glucosides due to rapid hydrolyzation to orellanine in the extract. Extraction with 3 M HCl or water mainly yielded orellanine, while MeOH or acidified MeOH mainly extracted mono- and diglucosylated orellanine. The highest recovery of total orellanine was obtained with 3 M HCl, which was subsequently used for quantitative analysis. A C18 HPLC column and low pH in the eluents retained all these toxins. Orellanine could be detected at a 4.9 ng/mL level in all extracts, which is well below the threshold for acute toxic effects. Additionally, the fragmentation pattern of orellanine upon electrospray MS/MS was probed. The method described is useful for two important applications. First, it allows quantitative analysis of processed food products that may be contaminated by orellanine from Cortinaceae mushrooms. Second, orellanine is currently being evaluated as a potential cure of metastatic renal cancer, and this work provides a method for monitoring orellanine at low concentrations within the therapeutic interval in blood serum.

A concept study on identification and attribution profiling of chemical threat agents using liquid chromatography-mass spectrometry applied to Amanita toxins in food.

Accidental or deliberate poisoning of food is of great national and international concern. Detecting and identifying potentially toxic agents in food is challenging due to their large chemical diversity and the complexity range of food matrices. A methodology is presented whereby toxic agents are identified and further characterized using a two-step approach. First, generic screening is performed by LC/MS/MS to detect toxins based on a list of selected potential chemical threat agents (CTAs). After identifying the CTAs, a second LC/MS analysis is performed applying accurate mass determination and the generation of an attribution profile. To demonstrate the potential of the methodology, toxins from the mushrooms Amanita phalloides and Amanita virosa were analyzed. These mushrooms are known to produce cyclic peptide toxins, which can be grouped into amatoxins, phallotoxins and virotoxins, where α-amanitin and ß-amanitin are regarded as the most potent. To represent a typical complex food sample, mushroom stews containing either A. phalloides or A. virosa were prepared. By combining the screening method with accurate mass analysis, the attribution profile for the identified toxins and related components in each stew was established and used to identify the mushroom species in question. In addition, the analytical data was consistent with the fact that the A. virosa specimens used in this study were of European origin. This adds an important piece of information that enables geographic attribution and strengthens the attribution profile.

Evaluation of a generic multi-analyte method for detection of >100 representative compounds correlated to emergency events in 19 food types by ultrahigh-pressure liquid chromatography-tandem mass spectrometry.

A generic extraction procedure combined with triple quadrupole mass spectrometric detection was evaluated for multi-residue analysis in 19 different foods. Measurable peaks could be obtained at relevant concentrations for 108 out of a total of 127 targeted compounds representing a wide range of physicochemical properties and compound classes related to emergency situations. Recoveries were determined for all 19 foods spiked with the 108 compounds. Seventy-five percent of the compounds had extraction recoveries of 70% or higher, with no compound below 46%. Suppression or enhancement effects on the MS response of the compounds dissolved in the extracts were low, as more than 80% of them had matrix effects between -35% and +20% and no compound was below -44% compared to matrix-free standard. In a validation, all compounds could be quantified at 200 µg/kg and 400 µg/kg food sample and 81% of the compounds at 40 µg/kg. It is concluded that the method is useful for the detection of various types of organic chemical toxicants at levels generally well below concentration thresholds for severe acute intoxication.

Cytotoxic potency of small macrocyclic knot proteins: structure-activity and mechanistic studies of native and chemically modified cyclotides.

The cyclotides are a family of circular and knotted proteins of natural origin with extreme enzymatic and thermal stability. They have a wide range of biological activities that make them promising tools for pharmaceutical and crop-protection applications. The cyclotides are divided into two subfamilies depending on the presence (Möbius) or absence (bracelet) of a cis-Pro peptide bond. In the current work we report a series of experiments to give further insight into the structure-activity relationship of cyclotides in general, and the differences between subfamilies and the role of their hydrophobic surface in particular. Selective chemical modifications of Glu, Arg, Lys and Trp residues was tested for cytotoxic activity: derivatives in which the Trp residue was modified showed low effect, demonstrating the existence of a connection between hydrophobicity and activity. However, over the full set of cyclotides examined, there was no strong correlation between the cytotoxic activity and their hydrophobicity. Instead, it seems more like that the distribution of charged and hydrophobic residues determines the ultimate degree of potency. Furthermore, we found that while the Glu residue is very important in maintaining the activity of the bracelet cyclotide cycloviolacin O2, it is much less important in the Möbius cyclotides. Despite these differences between cyclotide subfamilies, a systematic test of mixtures of cyclotides revealed that they act in an additive way.

Evaluation of toxicity and antitumor activity of cycloviolacin O2 in mice.

Cycloviolacin O2 is a small cyclic cysteine-rich protein belonging to the group of plant proteins called cyclotides. This cyclotide has been previously shown to exert cytotoxic activity against a variety of human tumor cell lines as well as primary cultures of human tumor cells in vitro. This study is the first evaluation of its tolerability and antitumor activity in vivo. Maximal-tolerated doses were estimated to 1.5 mg/kg for single intravenous (i.v.) dosing and 0.5 mg/kg for daily repeated dosing, respectively. Two different in vivo methods were used: the hollow fiber method with single dosing (i.v., 1.0 mg/kg) and traditional xenografts with repeated dosing over 2 weeks (i.v., 0.5 mg/kg daily, 5 days a week). The human tumor cell lines used displayed dose-dependent in vitro sensitivity (including growth in hollow fibers to confirm passage of cycloviolacin O2 through the polyvinylidene fluoride fibers), with IC5o values in the micromolar range. Despite this sensitivity in vitro, no significant antitumor effects were detected in vivo, neither with single dosing in the hollow fiber method nor with repeated dosing in xenografts. In summary, the results indicate that antitumor effects are minor or absent at tolerable (sublethal) doses, and cycloviolacin O2 has a very abrupt in vivo toxicity profile, with lethality after single injection at 2 mg/kg, but no signs of discomfort to the animals at 1.5 mg/kg. Repeated dosing of 1 mg/kg gave a local-inflammatory reaction at the site of injection after 2-3 days; lower doses were without complications.

Cyclotide proteins and precursors from the genus Gloeospermum: filling a blank spot in the cyclotide map of Violaceae.

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.

The conserved glu in the cyclotide cycloviolacin O2 has a key structural role.

Cyclotides are a large family of plant peptides that are characterised by a head-to-tail circular backbone and three disulfide bonds that are arranged in a cystine knot. This unique structural feature, which is referred to as a cyclic cystine knot, gives the cyclotides remarkable stability against chemical and biological degradation. In addition to their natural function as insecticides for plant defence, the cyclotides have a range of bioactivities with pharmaceutical relevance, including cytotoxicity against cancer cell lines. A glutamic acid residue, aside from the invariable disulfide array, is the most conserved feature throughout the cyclotide family, and it has recently been shown to be crucial for biological activity. Here we have used solution-state NMR spectroscopy to determine the three-dimensional structures of the potent cytotoxic cyclotide cycloviolacin O2, and an inactive analogue in which this conserved glutamic acid has been methylated. The structures of the peptides show that the glutamic acid has a key structural role in coordinating a set of hydrogen bonds in native cycloviolacin O2; this interaction is disrupted in the methylated analogue. The proposed mechanism of action of cyclotides is membrane disruption and these results suggest that the glutamic acid is linked to cyclotide function by stabilising the structure to allow efficient aggregation in membranes, rather than in a direct interaction with a target receptor.

The alpine violet, Viola biflora, is a rich source of cyclotides with potent cytotoxicity.

The cyclotides are currently the largest known family of head-to-tail cyclic proteins. The complex structure of these small plant proteins, which consist of approximately 30 amino acid residues, contains both a circular peptide backbone and a cystine knot, the combination of which produces the cyclic cystine knot motif. To date, cyclotides have been found in plants from the Rubiaceae, Violaceace and Cucurbitaceae families, and are believed to be part of the host defence system. In addition to their insecticidal effect, cyclotides have also been shown to be cytotoxic, anti-HIV, antimicrobial and haemolytic agents. In this study, we show that the alpine violet Viola biflora (Violaceae) is a rich source of cyclotides. The sequences of 11 cyclotides, vibi A-K, were determined by isolation and MS/MS sequencing of proteins and screening of a cDNA library of V. biflora in parallel. For the cDNA screening, a degenerate primer against a conserved (AAFALPA) motif in the cyclotide precursor ER signal sequence yielded a series of predicted cyclotide sequences that were correlated to those of the isolated proteins. There was an apparent discrepancy between the results of the two strategies as only one of the isolated proteins could be identified as a cDNA clone. Finally, to correlate amino acid sequence to cytotoxic potency, vibi D, E, G and H were analysed using a fluorometric microculture cytotoxicity assay using a lymphoma cell line. The IC(50)-values of the bracelet cyclotides vibi E, G and H ranged between 0.96 and 5.0 microM while the Möbius cyclotide vibi D was not cytotoxic at 30 microM.

Variations in cyclotide expression in viola species.

Cyclotides, a family of approximately 50 mini-proteins isolated from various Violaceae and Rubiaceae plants, are characterized by their circular peptide backbone and six conserved cysteine residues arranged in a cystine knot motif. Cyclotides show a wide range of biological activities, making them interesting targets for both pharmaceutical and agrochemical research, but little is known about their natural function and the events that trigger their expression. An investigation of the geographical and seasonal variations of cyclotide profiles has been performed, using the native Australian violet, Viola hederacea, and the Swedish sweet violet, Viola odorata, as model plants. The results showed that in the Australian violet the relative peptide levels of some cyclotides remained almost constant throughout the year, while other cyclotides were present only at certain times of the year. Therefore, it appears that V. hederacea expresses a basic armory of cyclotides as well as special "add-ons" whose levels are influenced by external factors. In the Swedish violet, cyclotide levels were increased up to 14 times during the warmest period of the year. The larger variation in expression levels of the Swedish plants may be a reflection of a greater climatic variation.