Fungal contaminants in Sicilian livestock feeds and first studies on the enzymatic activity of Aspergillus isolates

The purposes of this study were 1) to determine the total fungal contamination in Sicilian raw materials and livestock, 2) to evaluate the occurrence of Aspergillus spp., Penicillium spp. and Fusarium spp., 3) to identify fungi belonging to the genus Aspergillus and 4) to determine their ability to produce cellulolytic enzymes. Fourteen feed samples were collected in a feed mill near to Palermo (Sicily, Italy). Analysis of the total mycobiota was performed on Sabourad Dextros Agar (SAB) and Potato Dextrose Agar (PDA) and total fungal counts were expressed as CFU/g. Aspergillus spp. isolates were selected on the basis of the frequency of isolation and identifed using micro and macro-morphological characteristics and ITS sequence analysis. The ability of the Aspergillus isolates to produce cellulolytic enzymes was tested qualitatively by in vitro assay at two temperature, 25 and 30 °C, and in static and shaking condition. Total fungal population ranged from 1.11x106 to 1.31x108 and from 1.11x103 to 1.58x106 CFU/g on PDA and SAB, respectively. All feed samples showed the recurrent presence of colonies belonging mostly to the ubiquitous genera Aspergillus, Fusarium and Penicillium. Eight isolates of Aspergillus spp. were obtained and identifed as A. amstelodami, A. awamori, A. flavus, A. niger, A. oryzae and A. tubingensis. Between them, A. awamori, A. niger and A. tubingensis showed the highest enzymatic activity. The presence of potential mycotoxigenic isolates of Aspergillus spp. in the analysed feeds represents a risk for animal health; moreover their ability to produce cellulolytic enzymes can seriously affect feed quality

Computational methods to analyze and predict the binding mode of inhibitors targeting both human and mushroom tyrosinase

Tyrosinase, a copper-containing enzyme critical in melanin biosynthesis, is a key drug target for hyperpigmentation and melanoma in humans. Testing the inhibitory effects of compounds using tyrosinase from Agaricus bisporus (AbTYR) has been a common practice to identify potential therapeutics from synthetic and natural sources. However, structural diversity among human tyrosinase (hTYR) and AbTYR presents a challenge in developing drugs that are therapeutically effective. In this study, we combined retrospective and computational analyses with experimental data to provide insights into the development of new inhibitors targeting both hTYR and AbTYR. We observed contrasting effects of Thiamidol™ and our 4-(4-hydroxyphenyl)piperazin-1-yl-derivative (6) on both enzymes; based on this finding, we aimed to investigate their binding modes in hTYR and AbTYR to identify residues that significantly improve affinity. All the information led to the discovery of compound [4-(4-hydroxyphenyl)piperazin-1-yl](2-methoxyphenyl)methanone (MehT-3, 7), which showed comparable activity on AbTYR (IC50 = 3.52 μM) and hTYR (IC50 = 5.4 μM). Based on these achievements we propose the exploitation of our computational results to provide relevant structural information for the development of newer dual-targeting molecules, which could be preliminarily tested on AbTYR as a rapid and inexpensive screening procedure before being tested on hTYR

Evaluation of novel 4-(4-fluorobenzyl)piperazin-1-yl]-based compounds as competitive tyrosinase inhibitors endowed with anti-melanogenic effects

There is a considerable attention for the development of inhibitors of tyrosinase (TYR) as therapeutic strategy for the treatment of hyperpigmentation disorders in humans. Continuing in our efforts to identify TYR inhibitors, we describe the design, synthesis and pharmacophore exploration of new small molecules structurally characterized by the presence of the 4-fluorobenzylpiperazine moiety as key pharmacophoric feature for the inhibition of TYR from  Agaricus bisporus (AbTYR). Our investigations resulted in the discovery of the competitive inhibitor [4-(4-fluorobenzyl)piperazin-1-yl]-(3-chloro-2-nitro-phenyl)methanone 26 (IC 50  = 0.18 μM) that proved to be ∼100-fold more active than reference compound kojic acid (IC 50  = 17.76 μM). Notably, compound 26 exerted anti-melanogenic effect on B16F10 cells in absence of cytotoxicity. Docking analysis suggested its binding mode into AbTYR and into modelled human TYR

Design, Synthesis, and in Vitro Evaluation of 4-(4-Hydroxyphenyl)piperazine-Based Compounds Targeting Tyrosinase

Melanin biosynthesis is enzymatically regulated by tyrosinase (TYR, EC 1.14.18.1), which is efficiently inhibited by natural and synthetic phenols, demonstrating potential therapeutic application for the treatment of several human diseases. Herein we report the inhibitory effects of a series of (4-(4-hydroxyphenyl)piperazin-1-yl)arylmethanone derivatives, that were designed, synthesised and assayed against TYR from Agaricus bisporus (AbTYR). The best inhibitory activity was predominantly found for compounds bearing selected hydrophobic ortho-substituents on the aroyl moiety (IC50 values in the range of 1.5–4.6 μM). They proved to be more potent than the reference compound kojic acid (IC50=17.8 μM) and displayed competitive mechanism of inhibition of diphenolase activity of AbTYR. Docking simulation predicted their binding mode into the catalytic cavities of AbTYR and the modelled human TYR. In addition, these compounds displayed antioxidant activity combined with no cytotoxicity in MTT tests. Notably, the best inhibitor affected tyrosinase activity in α-MSH-stimulated B16F10 cells, thus demonstrating anti-melanogenic activity

Efficacy of ketamine in refractory convulsive status epilepticus in children: A protocol for a sequential design, multicentre, randomised, controlled, open-label, non-profit trial (KETASER01)

Introduction: Status epilepticus (SE) is a lifethreatening neurological emergency. SE lasting longer than 120 min and not responding to first-line and second-line antiepileptic drugs is defined as 'refractory' (RCSE) and requires intensive care unit treatment. There is currently neither evidence nor consensus to guide either the optimal choice of therapy or treatment goals for RCSE, which is generally treated with coma induction using conventional anaesthetics (high dose midazolam, thiopental and/or propofol). Increasing evidence indicates that ketamine (KE), a strong N-methyl-D-aspartate glutamate receptor antagonist, may be effective in treating RCSE. We hypothesised that intravenous KE is more efficacious and safer than conventional anaesthetics in treating RCSE. Methods and analysis: A multicentre, randomised, controlled, open-label, non-profit, sequentially designed study will be conducted to assess the efficacy of KE compared with conventional anaesthetics in the treatment of RCSE in children. 10 Italian centres/ hospitals are involved in enrolling 57 patients aged 1 month to 18 years with RCSE. Primary outcome is the resolution of SE up to 24 hours after withdrawal of therapy and is updated for each patient treated according to the sequential method. Ethics and dissemination: The study received ethical approval from the Tuscan Paediatric Ethics Committee (12/2015). The results of this study will be published in peer-reviewed journals and presented at international conferences

G.F.R. measuring with 99mTc-DTPA: limits in obstructive acute renal failure.

After a short introduction on the advantages and limits of G.F.R. determination with Ccr, the radioisotopic methods proposed by Schlegel and Gates for this parameter are described, and as well as ERPF and FF, without blood serial samples being necessary. In a severe renal failure case due to obstruction, the Authors have found a great mismatch in the results between the traditional methods and the radioisotopic ones. After the clinical case description, a possible explanation of this discrepancy is proposed: Schlegel and Gates' methods, which well correlate Ccr in chronic renal failure cases, in severe renal failure on obstructive basis, of recent onset, could not indicate the effective glomerular filtrate, but the nephronic mass, functionally blocked by the endocapsular hypertension secondary to the obstruction, but anatomically unaffected and so recoverable by a timely irradication of the obstruction

Cellulolytic activity in Aspergillus spp. contaminating livestock feeds and raw materials

The contamination by Aspergillus spp. have become a global concern in food and feedstuffs and can lead to a reduction in yield and quality of agricultural products with significant economic losses. Most species of Aspergillus produce cellulose-degrading enzymes and some of them also have mycotoxigenic activity. This study aimed i) to evaluate the Aspergillus contamination in feeds (16) and row materials (32) collected in Sicily; ii) to isolate and identify fungi belonging to the genus Aspergillus and iii) to analyze their ability to produce cellulolytic enzymes. Aspergillus spp. contamination was evaluated on PDA using serial ten-fold dilution and spread plate technique (Mirabile et al., 2019) and ranged from 50 to 9x106 CFU/g and from 45 to 3,3x107 in feeds and raw materials, respectively. The most recurrent colonies were identified by morphological features, ITS and β-tubulin sequence analysis as A. niger, A. tubingensis, A. brasiliensis, A. fumigatus and A. flavus. Qualitative production of cellulolytic enzymes performed according to Mandels et al. (1976) and time course of endo and exo-β-1,4 glucanase activity (UI/ml) determined in solid submerged fermentation (Ghose, 1987), revealed a variability between Aspergillus species and was strain-dependent. A. tubingensis SAAF14, A. flavus MUCL18903 and A. brasiliensis MUCL20039 exhibited the highest CMCase and FPase activity of 2.16, 2.37 and 0.99 UI/ml and 0.65, 0.92, and 0.42 UI/ml, respectively. The presence of these Aspergillus isolates with high cellulolytic activity could represent a potential risk for the quality of the contaminated food

Studio prospettico sulla ureterolitotomia laparoscopica: Approccio trans e retroperitoneale a confronto.

Studio prospettico sulla ureterolitotomia laparoscopica: Approccio trans e retroperitoneale a confronto

Leveraging the 3-Chloro-4-fluorophenyl Motif to Identify Inhibitors of Tyrosinase from <i>Agaricus bisporus</i>

Tyrosinase (EC 1.14.18.1) is implicated in melanin production in various organisms. There is a growing body of evidence suggesting that the overproduction of melanin might be related to several skin pigmentation disorders as well as neurodegenerative processes in Parkinson’s disease. Based on this consideration, the development of tyrosinase inhibitors represents a new challenge to identify new agents in pharmaceutical and cosmetic applications. With the goal of identifying tyrosinase inhibitors from a synthetic source, we employed a cheap and facile preliminary assay using tyrosinase from Agaricus bisporus (AbTYR). We have previously demonstrated that the 4-fluorobenzyl moiety might be effective in interactions with the catalytic site of AbTYR; moreover, the additional chlorine atom exerted beneficial effects in enhancing inhibitory activity. Therefore, we planned the synthesis of new small compounds in which we incorporated the 3-chloro-4-fluorophenyl fragment into distinct chemotypes that revealed the ability to establish profitable contact with the AbTYR catalytic site. Our results confirmed that the presence of this fragment is an important structural feature to improve the AbTYR inhibition in these new chemotypes as well. Furthermore, docking analysis supported the best activity of the selected studied compounds, possessing higher potency when compared with reference compounds