We describe the development and validation of a new quantitative real time PCR (qrt-PCR) method for the enumeration of the toxic benthic dinoflagellate Ostreopsis cf. ovata in marine environment. The benthic Ostreopsis sp. has a world-wide distribution and is associated during high biomass proliferation with the production of potent palytoxin-like compounds affecting human health and environment. Species-specific identification, which is relevant for the complex of different toxins production, by traditional methods of microscopy is difficult due to the high morphological variability, and thus different morphotypes can be easily misinterpreted. The method is based on the SYBR I Green real-time PCR technology and combines the use of a plasmid standard curve with a "gold standard" created with pooled crude extracts from environmental samples collected during a bloom event of Ostreopsis cf. ovata in the Mediterranean Sea. Based on their similar PCR efficiencies (95% and 98%, respectively), the exact rDNA copy number per cell was obtained in cultured and environmental samples. Cell lysates were used as the templates to obtain total recovery of DNA. The analytical sensitivity of the PCR was set at two rDNA copy number and 8.0×10(-4) cell per reaction for plasmid and gold standards, respectively; the sensitivity of the assay was of cells g(-1) fw or 1(-1) in macrophyte and seawater samples, respectively. The reproducibility was determined on the total linear quantification range of both curves confirming the accuracy of the technical set-up in the complete ranges of quantification over time. We developed a qrt-PCR assay specific, robust and high sample throughput for the absolute quantification of the toxic dinoflagellate Ostreopsis cf. ovata in the environmental samples. This molecular approach may be considered alternative to traditional microscopy and applied for the monitoring of benthic toxic microalgal species in the marine ecosystems.

The frequency and risk factors for intracerebral haemorrhage (ICH) after ischemic stroke are well-known. ICH frequency is increased by the use of antithrombotic or thrombolytic drugs. Several experimental studies have demonstrated a relationship between ICH and hypertension after fibrinolysis, but the optimal blood pressure levels in patients treated with recombinant tissue plasminogen activator (rTPA) are as yet unknown. We evaluated the role of blood pressure in patients with ischemic stroke treated with rTPA within 3h of symptom onset. We treated 86 consecutive patients admitted to our stroke unit between 2002 and 2008 and prospectively recorded the clinical and instrumental data in our stroke registry. We evaluated haemorrhagic complications by reviewing imaging findings. Blood pressure was recorded before rTPA and at 6, 12, 18, and 32h. Total cerebral haemorrhage occurred in eleven (12.7%) patients, and symptomatic intracerebral haemorrhage occurred in seven (8.1%). We failed to find a correlation between blood pressure levels and stroke severity at admission. High blood pressure levels correlated with a worse outcome. Systolic blood pressure was significantly higher in ICH patients relative to rTPA-treated patients without haemorrhagic complications (p<0.03). This study indicates that rTPA-induced haemorrhage is influenced by systolic blood pressure. More aggressive pharmacological reduction of hypertension during fibrinolysis and the subsequent 32h may reduce this complication.

Abstract The origin of endemic South American canid fauna has been traditionally linked with the rise of the Isthmus of Panama, suggesting that diversification of the dog fauna on this continent occurred very rapidly. Nevertheless, despite its obvious biogeographic appeal, the tempo of Canid evolution in South America has never been studied thoroughly. This issue can be suitably tackled with the inference of a molecular timescale. In this study, using a relaxed molecular clock method, we estimated that the most recent common ancestor of South American canids lived around 4 Ma, whereas all other splits within the clade occurred after the rise of the Panamanian land bridge. We suggest that the early diversification of the ancestors of the two main lineages of South American canids may have occurred in North America, before the Great American Interchange. Moreover, a concatenated morphological and molecular analysis put some extinct canid species well within the South American radiation, and shows that the dental adaptations to hypercarnivory evolved only once in the South American clade.

Aims: The ichthyotoxic species Prymnesium parvum (Haptophyceae) is difficult to quantify in a microscopy-based monitoring programme, because the cells are very small, fragile and their morphology can be distorted by the use of fixatives. In the attempt to overcome these problems, a real-time PCR-based method for the rapid and sensitive identification and quantification of P. parvum was developed. Methods and Results: A quantitative real-time PCR assay was optimized with primers designed on the internal transcribed spacer 2 rDNA region of P. parvum. This PCR assay was specific, showing no amplification of DNA extracted from closely related species, and sensitive. Moreover, this method was able to detect and reliably quantify P. parvum cells in preserved environmental samples artificially spiked with known amounts of cultured cells. Conclusions: Considering the specificity, sensitivity and applicability to preserved environmental samples, this method may be a useful tool for the monitoring of this toxic species. Significance and Impact of the Study: The real-time PCR method described in this study may represent a progress towards the rapid detection and quantification of P. parvum cells in water-monitoring programmes, allowing the early application of strategies to control bloom events, such as the use of clay minerals.

Recent studies have suggested that abnormalities of dopamine and trace amines (tyramine, octopamine, and synephrine), products of tyrosine metabolism, may constitute the metabolic events that predispose to the occurrence of cluster headache (CH) and migraine attacks. This hypothesis is supported by the following evidences: the discovery of trace amine associated receptors (TAARs), expressed on the olfactory epithelium, amigdala, hypothalamus, periacqueductal gray, and the biochemical anomalies of dopamine and trace amines. The possible effects of these biochemical abnormalities on TAARs and dopamine receptors, located in different areas of CNS, may explain the behaviour (restlessness, anxiety and, at times, hypersexuality) and the autonomic signs during the painful attacks of CH, and the premonitory symptoms of migraine crisis (thirst, craving, yawning, alteration of smell, depression etc.).

AIDA Cefalee is a database for the management of headache patients developed on behalf of the Italian Neurological Association for Headache Research (ANIRCEF). The system integrates a diagnostic expert system able to suggest the correct ICHD-II diagnosis once all clinical characteristics of a patient's headache have been collected. The software has undergone a multicentre validation study to assess: its diagnostic accuracy; the impact of using the software on visit duration; the userfriendliness degree of the software interface; and patients' acceptability of computer-assisted interview. Five Italian headache centres participated in the study. The results of this study validate AIDA Cefalee as a reliable diagnostic tool for primary headaches that can improve diagnostic accuracy with respect to the standard clinical method without increasing the time length of visits even when used by operators with basic computer experience.

Trace amines, including tyramine, octopamine and synephrine, are closely related to classic biogenic amines. In one study, where these substances were found elevated in plasma of migraineurs, it was hypothesized that trace amine metabolism is deranged in migraine. To confirm these findings, we studied, using a multichannel electrochemical high-performance liquid chromatography system, the concentrations of trace amines in platelets of migraine without aura (MoA) and migraine with aura (MA) patients in headache-free period, compared with controls. Platelet concentrations of trace amines, although elevated in both migraine types, showed a different profile in MoA and MA. Octopamine was significantly higher in MoA sufferers (0.69 +/- 0.43 ng/10(8) platelets) compared with both control subjects (0.22 +/- 0.16 ng/10(8) platelets) and MA patients (0.39 +/- 0.37 ng/10(8) platelets). Synephrine was significantly higher in MA patients (0.72 +/- 0.44 ng/10(8) platelets) with respect to both controls (0.33 +/- 0.25 ng/10(8) platelets) and MoA sufferers (0.37 +/- 0.29 ng/10(8) platelets). These results strengthen the hypothesis that tyrosine metabolism is deranged in migraine and may participate in its pathophysiology.

Hot dogs contain apparent N-nitroso compounds (ANC) and ANC precursors (ANCP). ANCP purification was followed by nitrosation, sulfamic acid treatment, and analysis for ANC. Aqueous hot dog extracts were adsorbed on silica gel, which was eluted with MeCN and MeOH. The MeOH eluate was adsorbed on cation exchange resin (H(+) form) and eluted with NH(4)OH. Eluted ANCP traveled at moderate speeds in high-performance liquid chromatography (HPLC) on amino and Pb(2+) columns. Gas chromatography-mass spectrometry (GC-MS) of trimethylsilyl (TMS) derivatives of crude water extract indicated the presence of glycerol, phosphate, lactic acid, and two monosaccharides. GC-MS of TMS derivatives of Pb(2+) column HPLC eluates indicated that ANCP included 1-deoxy-N-1-glucosyl glycine. The nitrosated NH(4)OH eluate showed 4x background mutagenic activity for Salmonella typhimurium TA-100. Un-nitrosated fractions showed 2x background activity. Although tryptophan nitrosation gave 88% ANC yield, tryptophan is probably not a major ANCP in hot dogs. Hot dog patties prepared with or without sucrose or glucose showed similar ANC and ANCP levels. We discuss possible implications of these findings for the etiology of colon cancer.

Objective.-To measure plasma and platelet levels of dopamine in patients with migraine with aura, migraine without aura, and cluster headache. Background.-Clinical, genetic, and pharmacological evidences suggest that an abnormality of dopaminergic system plays a role in migraine pathogenesis. Direct evidence of an abnormal metabolism of dopamine in migraine, however, is lacking. Methods.-Plasma and platelet levels of dopamine were measured in patients with migraine with aura or migraine without aura during headache-free periods and in patients with cluster headache during the remission and active periods, as compared with healthy control subjects, using a multichannel electrochemical high-performance liquid chromatography system. Results.-Plasma levels of dopamine were not detectable with our methodology. Platelet levels of dopamine were higher in both types of migraine (migraine without aura =.20 +/-.17 ng/10(8) platelets; migraine with aura =.16 +/-.19 ng/10(8) platelets) than in control subjects (.10 +/-.11 ng/10(8) platelets), although in migraine with aura patients the difference was not significant. Patients with cluster headache showed the highest levels of platelet dopamine (.34 +/-.36 ng/10(8) platelets). Conclusions.-Our results support the hypothesis that the dopaminergic system is impaired in migraine and cluster headache and suggest that high platelet levels of dopamine may represent an abnormal biochemical phenotypic trait of these primary headaches.(Headache 2006;46:585-591).