Complexing the Marine Sesquiterpene Euplotin C by Means of Cyclodextrin-Based Nanosponges: A Preliminary Investigation

Euplotin C is a sesquiterpene of marine origin endowed with significant anti-microbial and anti-tumor properties. Despite the promising functional profile, its progress as a novel drug candidate has failed so far, due to its scarce solubility and poor stability in aqueous media, such as biological fluids. Therefore, overcoming these limits is an intriguing challenge for the scientific community. In this work, we synthesized β-cyclodextrin-based nanosponges and investigated their use as colloidal carriers for stably complex euplotin C. Results obtained proved the ability of the carrier to include the natural compound, showing remarkable values of both loading efficiency and capacity. Moreover, it also allowed us to preserve the chemical structure of the loaded compound, which was recovered unaltered once extracted from the complex. Therefore, the use of β-cyclodextrin-based nanosponges represents a viable option to vehiculate euplotin C, thus opening up its possible use as pharmacologically active compound.     

Selection,characterization and genetic analysis of laboratory mutants of <Emphasis Type="Italic">Botryotinia fuckeliana</Emphasis> (<Emphasis Type="Italic">Botrytis cinerea</Emphasis>) resistant to the fungicide boscalid

Resistance to the fungicide boscalid in laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) was investigated. The baseline sensitivity to boscalid was evaluated in terms of colony growth (EC₅₀ = 0.3–3 μg ml⁻¹; MIC = 10–30 μg ml⁻¹) and conidial germination (EC₅₀ = 0.03–0.1 μg ml⁻¹; MIC = 1–3 μg ml⁻¹) tests. Mutants were selected in vitro from wild-type strains of the fungus on a fungicide-amended medium containing acetate as a carbon source. Mutants showed two different levels of resistance to boscalid, distinguishable through the conidial germination tests: low (EC₅₀ ∼ 0.3 μg ml⁻¹, ranging from 0.03 to 1 μg ml⁻¹; MIC > 100 μg ml⁻¹) and high (EC₅₀ > 100 μg ml⁻¹) resistance. Analysis of meiotic progeny from crosses between resistant mutants and sensitive reference strains showed that resistant phenotypes were due to mutations in single major gene(s) inherited in a Mendelian fashion, and linked with both the Daf1 and Mbc1 genes, responsible for resistance to dicarboximide and benzimidazole fungicides, respectively. Gene sequence analysis of the four sub-units of the boscalid-target protein, the succinate dehydrogenase enzyme, revealed that single or double point mutations in the highly conserved regions of the iron-sulphur protein (Ip) gene were associated with resistance. Mutations resulted in proline to leucine or phenylalanine replacements at position 225 (P225L or P225F) in high resistant mutants, and in a histidine to tyrosine replacement at position 272 (H272Y) in low resistant mutants. Sequences of the flavoprotein and the two transmembrane sub-units of succinate dehydrogenase were never affected.     

Three cases of prolapse of the nictitans gland in cats

We report three cases of adult cats showing a prolapse of the gland of the third eyelid. Three different breeds were affected: Burmese, Persian and Domestic Short-haired. In all cases, the disorder occurred spontaneously, without any other ocular sign. Surgical replacement of the gland was performed using the Morgan pocket technique. Good esthetic results were obtained, and no recurrence occurred.     

Treatment of cereal products with a tailored preparation of trichoderma enzymes increases the amount of soluble dietary fiber

Nutritionists recommend increasing the intake of soluble dietary fiber (SDF), which is very low in most cereal-based products. Conversion of insoluble DF (IDF) into SDF can be achieved by chemical treatments, but this affects the sensorial properties of the products. In this study, the possibility of getting a substantial increase of SDF from cereal products using a tailored preparation of Trichoderma enzymes is reported. Enzymes were produced cultivating Trichoderma using durum wheat fiber (DWF) and barley spent grain (BSG) as unique carbon sources. Many Trichoderma strains were screened, and the hydrolysis conditions able to increase by enzymatic treatment the amount of SDF in DWF and BSG were determined. Results demonstrate in both products that it is possible to triple the amount of SDF without a marked decrease of total DF. The enzymatic treatment also causes the release of hydroxycinnamic acids, mainly ferulic acid, that are linked to the polysaccharides chains. This increases the free phenolic concentration, the water-soluble antioxidant activity, and, in turn, the phenol compounds bioavailability.     

Genetic analysis and molecular characterisation of laboratory and field mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to QoI fungicides

BACKGROUND: QoI fungicides, inhibitors of mitochondrial respiration, are considered to be at high risk of resistance development. In several phytopathogenic fungi, resistance is caused by mutations (most frequently G143A) in the mitochondrial cytochrome b (cytb) gene. The genetic and molecular basis of QoI resistance were investigated in laboratory and field mutants of Botryotinia fuckeliana (de Bary) Whetz. exhibiting in vitro reduced sensitivity to trifloxystrobin. RESULTS: B. fuckeliana mutants highly resistant to trifloxystrobin were obtained in the laboratory by spontaneous mutations in wild‐type strains, or from naturally infected plants on a medium amended with 1–3 mg L^?1 trifloxystrobin and 2 mM salicylhydroxamic acid, an inhibitor of alternative oxidase. No point mutations were detected, either in the complete nucleotide sequences of the cytb gene or in those of the aox and Rieske protein genes of laboratory mutants, whereas all field mutants carried the G143A mutation in the mitochondrial cytb gene. QoI resistance was always maternally inherited in ascospore progeny of sexual crosses of field mutants with sensitive reference strains. CONCLUSIONS: The G143A mutation in cytb gene is confirmed to be responsible for field resistance to QoIs in B. fuckeliana. Maternal inheritance of resistance to QoIs in progeny of sexual crosses confirmed that it is caused by extranuclear genetic determinants. In laboratory mutants the heteroplasmic state of mutated mitochondria could likely hamper the G143A detection, otherwise other gene(s) underlying different mechanisms of resistance could be involved. Copyright © 2012 Society of Chemical Industry     

Towards a rapid molecular identification of the common phlebotomine sand flies in the Mediterranean region

The present study reports two simple molecular approaches allowing a rapid identification of the most prevalent species of phlebotomine sand flies in the Mediterranean region. A PCR protocol for the amplification of ITS2 ribosomal region and a PCR-RFLP on a mitochondrial DNA fragment (cytb-nd1) were settled in order to identify and discriminate among Phlebotomus perniciosus, Phlebotomus neglectus, Phlebotomus perfiliewi, Phlebotomus papatasi and Sergentomyia minuta. The ITS2 regions showed a certain degree of interspecific variability, which led to PCR amplicons of different sizes, i.e., 450, 490, 460, 480 and 530 bp for P. perniciosus, P. neglectus, P. perfiliewi, P. papatasi, and S. minuta, respectively. Analogously, the digestion of a mitochondrial DNA amplicon with Ase I enzyme showed five different restriction profiles, which allowed the unequivocal differentiation of the sand fly species examined. These methods might represent useful tools for a molecular large scale screening of phlebotomine sand fly species caught in areas where leishmaniasis is endemic, in order to plan appropriate epidemiological surveillance programs for both Leishmania spp. and their vectors.     

Detection of Mycobacterium avium subsp. paratuberculosis by buoyant density centrifugation, sequence capture PCR and dot blot hybridisation

Detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by polymerase chain reaction (PCR) is often hampered by the lack of efficient methods for sample treatment. We report a protocol for analysis of faecal samples based on buoyant density centrifugation in Percoll and IS900 sequence capture PCR combined with a dot blot assay for detection of low-grade infection of M. paratuberculosis. Serial dilutions of M. paratuberculosis genomic DNA and M. paratuberculosis bacteria were used to assess the sensitivity of the method. The final evaluation was performed with spiked faecal samples, which also were analysed by culture. The presence of PCR inhibitory substances in processed faecal samples was evaluated by including a PCR internal control. By using buoyant density centrifugation, sequence capture PCR, and dot blot hybridisation, we achieved a sensitivity of 10(3)CFU (colony forming units)/g of faeces. The detection limit by culture was assessed to 10(2)CFU/g of faeces. We conclude that the described protocol is a fast and sensitive alternative to bacterial culture of faecal samples.     

Genetic analysis and phenotypic associations for drought tolerance in Hordeum spontaneum introgression lines using SSR and SNP markers

Associations between markers and drought related traits were investigated on a set of 57 advanced barley breeding lines, carrying various levels of introgression from Hordeum spontaneum lines 41-1 and 41-5, the best sources of drought tolerance in the ICARDA barley breeding program, using 74 simple sequences repeats (SSR) and 20 single nucleotide polymorphism markers. The 57 lines were evaluated for grain yield and drought related traits for three years (2003/04, 2004/05, 2005/06) in nine Mediterranean low rainfall environments. A high level of polymorphism was found with SSR markers, and the mean polymorphism information content and gene diversity values were 0.67 and 0.71, respectively. The number of alleles per locus varied from 2 to 11, with an average of 5.8 alleles per marker. Considering all the 57 lines, the linkage disequilibrium (LD) analysis was significant at a comparison-wise P?<?0.01 level in nearly 9 % of the SSR marker pairs used and a decay of LD was observed to a value of r ²?<?0.2 at a genetic distance of 40?cM. The association analysis revealed a total of 147 significant marker?Ctrait associations for grain yield and drought related traits. A total of 72 (49 %) marker?Ctrait associations showed favorable effects of the exotic germplasm where the H. spontaneum lines contributed to an improvement of the trait under drought stress conditions. The number of significant marker?Ctrait associations per trait were: 12 for growth habit; 2 for growth vigor; 11 for peduncle extrusion; 5 for number of grains per spike; 20 for peduncle length; 16 for days to heading; 20 for plant height; 8 for spike length; 17 for thousand kernel weight; 30 for grain yield; 4 for harvest index and 2 for biological yield. The phenotypic variation explained by individual marker?Ctrait associations ranged from 7.6 % to 36.2 %. The identification of genomic regions associated with grain yield and drought related traits is useful for the genetic improvement of cultivars better adapted to drought stress environments. Thus, the present study is encouraging in identifying significant marker?Ctrait associations through LD based association mapping analysis, which could complement and augment previous quantitative trait loci information for the potential use of Marker Assisted Selection for drought.     

TRAP molecular markers as a system for saturation of the genetic map of durum wheat

Target region amplification polymorphism (TRAP) is a relatively new PCR-based technique that detects large numbers of loci in a single reaction without extensive pre-PCR processing of samples. The aim of this study was to integrate TRAP markers in an EST-derived SSR linkage map of a RIL mapping population from the cross of the durum wheat cultivars Ciccio and Svevo, for a more general purpose of establishing a high-throughput system for genetic map saturation. Primer combinations producing PCR products with at least 4–5 polymorphic bands were selected and analyzed across the mapping population. The PCR reactions produced a total of 2,881 fragments with an average of 52 peaks per reaction. A total of 142 new TRAP markers were mapped and found to be randomly distributed in the genome. The total length of the map was 2,043.0 cM, with an average chromosome length of 145.9 cM. Homoeologous group one had the highest number of TRAP markers (38 loci) and the longest map length (407.9 cM) for a total of 87 markers, while the homoeologous group five had the lowest TRAP marker number (5 loci) and the shortest map length (232.5 cM). The distribution of markers among the seven homoeologous groups was random. The results indicate that TRAP is highly efficient in genetic mapping, generating a large number of markers scattered across the genome. This closes many existing gaps in marker coverage and may join otherwise separate linkage groups.