Assunta Zanfini

Quantification of psychosine in the serum of twitcher mouse by LC-ESI-tandem-MS analysis.

Globoid cell leukodystrophy or Krabbe disease is an inherited autosomal recessive disorder caused by mutations in the galactosylceramidase (GALC) gene. Deficiency of GALC results in the accumulation of a highly cytotoxic metabolite galactosylsphingosine (psychosine). In the present study, we describe the development and validation of a sensitive and specific LC-ESI-tandem-MS method for the determination of psychosine in the serum of twitcher mice, the naturally occurring animal model of this disease. The method was validated in terms of accuracy, precision, specificity, linearity and sensitivity. Calibration plots were linear over the concentration range of 2.5-50ng/mL. Recovery of psychosine from serum was in the range 94.20-98.02%. The results of this study show that in the affected mice the concentration of psychosine (ranging from 2.53 to 33.27ng/mL) increased significantly with the progression of the disease. The maximum level (33.27ng/mL) was detected in the serum of one of the twitcher mice sacrificed at 40PND. Psychosine was not detected at significant levels in wild type mice. These results clearly demonstrate that this noninvasive, rapid, and highly sensitive LC-ESI-tandem-MS method is a very useful approach for the detection of psychosine in serum.

THE POTENTIAL BENEFICIAL EFFECTS OF TENEBRIO MOLITOR (COLEOPTERA TENEBRIONIDAE) AND GALLERIA MELLONELLA (LEPIDOPTERA PYRALIDAE) ON HUMAN HEALTH

(*) Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria Research Centre for Plant Protection and Certification (CREA-DC), via Lanciola 12/A, 50125 Firenze, Italy. (**) Department of Biotechnology, Chemistry and Pharmacy, University of Siena, via A. Moro, 53100 Siena, Italy. (***) Lead Discovery Siena Srl, via Vittorio Alfieri 31, 53019 Castelnuovo Berardenga Siena, Italy. Corresponding author: annaritacito@yahoo.it

Cuticle-degrading proteases and toxins as virulence markers of Beauveria bassiana (Balsamo) Vuillemin.

Beauveria bassiana is one of the most known entomopathogenic fungal species and its entomopathogenic mechanism involves several bioactive metabolites, mainly cuticle‐degrading enzymes and toxic molecules, which are predicted to play a key role as virulence factors. In this study six Beauveria bassiana strains (B 13/I03, B 13/I11, B 13/I49, B 13/I57, B 13/I63, and B 13/I64) were assayed against Tenebrio molitor larvae. Enzymatic activity of total proteases and specifically Pr 1 and Pr 2, as well as the production of toxic compounds were investigated in each fungal strain. Toxins were detected both in vitro—in medium filtrates and mycelia—and in vivo—in Tenebrio molitor larvae infected by the fungal strains tested. B 13/I11 and B 13/I63 strains showed the most significant entomopathogenic activity against Tenebrio molitor larvae (cumulative mortality rate 100 and 97%, respectively; average survival time 5.85 and 6.74 days, respectively). A widely variable and fungal strain‐dependent enzymatic activity of total proteases, Pr 1 and Pr 2 was found. Beauvericin, beauvericin A and bassianolide resulted the most prevalent toxins detected in the substrates analyzed. It has been found that an increase of beauvericin content in vivo resulted significantly correlated to a decrease of Tenebrio molitor larvae average survival time in entomopathogenic bioassay (inverse correlation). The involvement of beauvericin in B. bassiana entomopathogenic process is confirmed; in vitro analysis of cuticle degrading proteases activity and toxins production in relation to the methods adopted resulted insufficient for a rapid screening to determine the virulence of B. bassiana strains against Tenebrio molitor larvae.