Anatoxin-a producing Tychonema (Cyanobacteria) in European waterbodies.

In order to identify the cyanobacterial species responsible of anatoxin-a (ATX) production in Lake Garda (Northern Italy), an intensive isolation and culturing of filamentous cyanobacteria were established since 2014 from environmental samples. In this work, we report a detailed account of the strategy adopted, which led to the discovery of a new unexpected producer of ATX, Tychonema bourrellyi. So far, this species is the first documented example of cultured Oscillatoriales able to produce ATX isolated from pelagic freshwater ecosystems. The isolated filaments were identified adopting a polyphasic approach, which included microscopic species identification, genetic characterisation and phylogenetic analyses based on 16S rRNA genes. The taxonomic identification was further confirmed by the high (>99%) rbcLX sequence similarities of the T. bourrellyi strains of Lake Garda with those deposited in DNA sequence databases. More than half of the isolates were shown to produce a significant amount of ATX, with cell quota ranging between 0.1 and 2.6 µg mm(-3), and 0.01 and 0.35 pg cell(-1). The toxic isolates were tested positive for anaC of the anatoxin-a synthetase (ana) gene cluster. These findings were confirmed with the discovery of one ATX producing T. bourrellyi strain isolated in Norway. This strain and a further non-ATX producing Norwegian Tychonema bornetii strain tested positive for the presence of the anaF gene of the ana gene cluster. Conversely, none of the Italian and Norwegian Tychonema strains were positive for microcystins (MCs), which was also confirmed by the absence of mcyE PCR products in all the samples analysed. This work suggests that the only reliable strategy to identify cyanotoxins producers should be based on the isolation of strains and their identification with a polyphasic approach associated to a concurrent metabolomic profiling.

Antiherpetic efficacy of aqueous extracts of the cyanobacterium Arthrospira fusiformis from Chad.

Natural substances offer interesting pharmacological perspectives for antiviral drug development with regard to broad spectrum antiviral properties and novel modes of action. Drugs currently used to treat cutaneous or genital herpetic infections are effective in limiting disease, but the emergence of drug-resistant viruses in immunocompromised individuals can be problematic. A nontoxic cyanobacterium Arthrospira strain from Chad has been characterized by sequence analysis of the intergenic spacer region of the phycocyanin gene. This cyanobacterium was identified as Arthrospira fusiformis by phylogenetic tree analysis. The antiherpetic activity of crude aqueous extracts from the Chad A. fusiformis isolate was determined. Antiviral efficacy against herpes simplex virus of cold water extract, hot water extract and phosphate buffer extract was assessed in plaque reduction assays and their mode of antiherpetic action was analysed. In virus suspension assays, cold water extract, hot water extract and phosphate buffer extract inhibited virus infectivity by 54.9%, 64.6%, and 99.8%, respectively, in a dose-dependent manner. The mode of antiviral action was determined by addition of cyanobacterial extracts separately at different time periods during the viral infection cycle. Extracts of A. fusiformis strain clearly inhibited herpesvirus multiplication before and during virus infection of host cells. The phosphate buffer extract of the A. fusiformis strain affected free herpes simplex virus prior to infection of host cells and inhibited intracellular viral replication. It is concluded, that Arthrospira compounds warrant further investigation to examine their potential role in the treatment of herpetic infections.

Molecular authentication and characterization of the antiherpetic activity of the cyanobacterium Arthrospira fusiformis.

In recent years there has been an increasing interest for application of natural products as antiinfectives and concerns about the safety of synthetic compounds have encouraged more detailed studies of natural resources. Two different strains of the nontoxic cyanobacterium Arthrospira from the United States and Egypt have been characterized by sequence analysis of the intergenic spacer region of the phycocyanin gene. Both cyanobacteria were identified as Arthrospira fusiformis by phylogenetic tree analysis. The antiherpetic activity of crude aqueous extracts from the US and the Egyptian A. fusiformis isolates was determined. Antiviral activity against herpes simplex virus of cold water extracts, hot water extracts and phosphate buffer extracts from the American and the Egyptian strains was assessed in plaque reduction assays and their mode of antiherpetic action was analysed. In virus suspension assays, all extracts of the American cyanobacterium and the phosphate buffer extract of the Egyptian cyanobacterium inhibited virus infectivity by > 90% in a dose-dependent manner. Phosphate buffer extract and hot water extract of the US cyanobacterium demonstrated the highest antiviral activity at low extract concentrations with high selectivity indices of 7464 and 542, respectively. The mode of antiviral action has been determined by addition of cyanobacterial extracts separately at different time periods during the viral infection cycle. Two extracts of the US A. fusiformis strain clearly inhibited herpesvirus multiplication before and after virus infection of host cells. In contrast, extracts of the Egyptian A. fusiformis strain affected only free herpes simplex virus prior to infection of host cells by direct inactivation of virus particles. In this study different Arthrospira crude extracts showed a significant antiviral effect and might be applied in recurrent herpetic infections.

A rapid quantitative determination of triglycerides by means of thin-layer chromatography.

A quantitative chromatographic method for determining triglycerides in serum is described. The serum is applied directly to a thin-layer plate and after partition with one developing solvent the fractions are charred with 10% sulphuric acid. Charring takes 20 minutes. The concentration of triglycerides is measured against a triolein standard, which in itself is corrected for having too many double bonds by scanning the charring of the triglyceride fraction with a "flying spot"-scanner or a slitscanner. The results are compared with other methods of determination.