Immobilization of myrosinase (Thioglucoside glucohydrolase EC 3.2.3.1)

Summary Myrosinase, purified from white mustard seeds by affinity chromatography and homogeneous in SDS-PAGE, was used in immobilization experiments on common inexpensive solid matrices viz. trimaleylchitosan, gamma-alumina, gamma-alumina activated by dodecadiamin, silanized sand, silanized silica and cellulose triacetate. For each matrix the absorption and immobilization yields at pHs 5.5 and 7.2 were determined. For these parameters, gamma-alumina showed the most interesting results. The maximum amount of enzyme immobilized was ca. 30 mg/g of gamma-alumina. The enzyme also showed good static and operational stability.     

The life cycle of <Emphasis Type="Italic">Amblyomma auricularium</Emphasis> (Acari: Ixodidae) using rabbits (<Emphasis Type="Italic">Oryctolagus cuniculus</Emphasis>) as experimental host

The life cycle of Amblyomma auricularium (Conil) is reported for the first time, using rabbits as experimental host. Developmental periods of free-living stages were observed in an incubator at 27 ± 1°C, 80 ± 10% RH and 24 h darkness. The complete life cycle, including pre-feeding periods for each parasitic stage, ranged from 97 to 162 days. The overall sex ratio was 1.16:1 (M:F). Feeding and premolt periods, molting success, and engorgement weight of nymphs were statistically different between males and females (P < 0.01), but because their ranges overlapped, they cannot be used to predict the sex with accuracy. The potential role of rabbits as experimental hosts for rearing A. auricularium in the laboratory is discussed.     

Trichoderma spp. tolerance to Brassica carinata seed meal for a combined use in biofumigation

Biofumigation by Brassicaceae green manure or seed meal incorporation into soil is an ecological alternative to chemical fumigation against soil-borne pathogens, based on the release of glucosinolate-derived compounds. This study aimed at investigating the tolerance of the beneficial fungus Trichoderma to these compounds in view to combined utilization with Brassica carinata seed meal (BCSM). Forty isolates of Trichoderma spp. were tested in vitro for tolerance to toxic volatiles released by BCSM and in direct contact with the meal. They were found to be generally less sensitive than the assayed pathogens (Pythium ultimum, Rhizoctonia solani, Fusarium oxysporum), even if a fungistatic effect was observed at the highest dose (10 μmole of sinigrin). Most of them also were able to grow on BCSM and over the pathogens tested. A preliminary experiment of integrating BCSM with Trichoderma in soil was carried out under controlled conditions with the patho-system P. ultimum—sugar beet. BCSM incorporation increased pathogen population, but reduced disease incidence, probably due to indirect mechanisms. The greatest effect was achieved when BCSM was applied in combination with Trichoderma, regardless of meal ability to release isothiocyanate. These findings suggest that disease control can be improved by this integrated approach. This study also highlighted that a reduction of allyl-isothiocyanate concentration in soil could occur due to the activity of some Trichoderma isolates. This effect could protect resident or introduced Trichoderma isolates from depressing effects due to the biocidal compounds, but, on the other hand, could reduce the efficacy of biofumigation against target pathogens.     

Candida spp. morphotype differentiation on Sabouraud-Triphenyltetrazolium-Agar (STTZ-Agar) under three different experimental conditions

One hundred and thirty-two strains of Candida spp. were cultured on STTZ-Agar at 37 degrees C for 6 days and at 25 degrees C for 6 and 21 days to determine the culture conditions that would ensure maximum reproducibility in the discrimination of the strains of the same species. Standardization is of utmost importance, as varying experimental conditions can alter the results of the tests. Further studies are needed also implementing molecular tests to establish possible relationships between morphotype, genotype and virulence.     

Comprehensive mapping of cystic fibrosis mutations to CFTR protein identifies mutation clusters and molecular docking predicts corrector binding site

Cystic Fibrosis (CF) is caused by mutations in the CFTR gene, of which over 2000 have been reported to date. Mutations have yet to be analyzed in aggregate to assess their distribution across the tertiary structure of the CFTR protein, an approach that could provide valuable insights into the structure‐function relationship of CFTR. In addition, the binding site of Class I correctors (VX‐809, VX‐661, and C18) is not well understood. In this study, exonic CFTR mutations and mutant allele frequencies described in 3 curated databases (ABCMdb, CFTR1, and CFTR2, comprising >130 000 data points) were mapped to 2 different structural models: a homology model of full‐length CFTR protein in the open‐channel state, and a cryo‐electron microscopy core‐structure of CFTR in the closed‐channel state. Accordingly, residue positions of 6 high‐frequency mutant CFTR alleles were found to spatially co‐localize in CFTR protein, and a significant cluster was identified at the NBD1:ICL4 interdomain interface. In addition, immunoblotting confirmed the approximate binding site of Class I correctors, demonstrating that these small molecules act via a similar mechanism in vitro, and in silico molecular docking generated binding poses for their complex with the cryo‐electron microscopy structure to suggest the putative corrector binding site is a multi‐domain pocket near residues F374‐L375. These results confirm the significance of interdomain interfaces as susceptible to disruptive mutation, and identify a putative corrector binding site. The structural pharmacogenomics approach of mapping mutation databases to protein models shows promise for facilitating drug discovery and personalized medicine for monogenetic diseases.     

Development of a new wheat microarray from a durum wheat totipotent cDNA library used for a powdery mildew resistance study

Totipotent cDNA libraries representative of all the potentially expressed sequences in a genome would be of great benefit to gene expression studies. Here, we report on an innovative method for creating such a library for durum wheat (Triticum turgidum L. var. durum) and its application for gene discovery. The use of suitable quantities of 5-azacytidine during the germination phase induced the demethylation of total DNA, and the resulting seedlings potentially express all of the genes present in the genome. A new wheat microarray consisting of 4925 unigenes was developed from the totipotent cDNA library and used to screen for genes that may contribute to differences in the disease resistance of two near-isogenic lines, the durum wheat cultivar Latino and the line 5BIL-42, which are respectively susceptible and resistant to powdery mildew. Fluorescently labeled cDNA was prepared from the RNA of seedlings of the two near-isogenic wheat lines after infection with a single powdery mildew isolate under controlled conditions in the greenhouse. Hybridization to the microarray identified six genes that were differently expressed in the two lines. Four of the sequences could be assigned putative functions based on their similarity to known genes in public databases. Physical mapping of the six genes localized them to two regions of the genome: the centromeric region of chromosome 5B, where the Pm36 resistance gene was previously localized, and chromosome 6B.     

An isotopic method for testing the influence of leaf litter quality on carbon fluxes during decomposition

During microbial breakdown of leaf litter a fraction of the C lost by the litter is not released to the atmosphere as CO₂ but remains in the soil as microbial byproducts. The amount of this fraction and the factors influencing its size are not yet clearly known. We performed a laboratory experiment to quantify the flow of C from decaying litter into the soil, by means of stable C isotopes, and tested its dependence on litter chemical properties. Three sets of ¹³C-depleted leaf litter (Liquidambar styraciflua L., Cercis canadensis L. and Pinus taeda L.) were incubated in the laboratory in jars containing ¹³C-enriched soil (i.e. formed C4 vegetation). Four jars containing soil only were used as a control. Litter chemical properties were measured using thermogravimetry (Tg) and pyrolysis–gas chromatography/mass spectrometry–combustion interface–isotope ratio mass spectrometry (Py–GC/MS–C–IRMS). The respiration rates and the δ¹³C of the respired CO₂ were measured at regular intervals. After 8 months of incubation, soils incubated with both L. styraciflua and C. canadensis showed a significant change in δ¹³C (δ¹³C_final = −20.2 ± 0.4‰ and −19.5 ± 0.5‰, respectively) with respect to the initial value (δ¹³C_initial = −17.7 ± 0.3‰); the same did not hold for soil incubated with P. taeda (δ¹³C_final:−18.1 ± 0.5‰). The percentages of litter-derived C in soil over the total C loss were not statistically different from one litter species to another. This suggests that there is no dependence of the percentage of C input into the soil (over the total C loss) on litter quality and that the fractional loss of leaf litter C is dependent only on the microbial assimilation efficiency. The percentage of litter-derived C in soil was estimated to be 13 ± 3% of total C loss.     

Relationship between aerobic fitness and metabolic power metrics in elite male soccer players

The aim was to assess the relationship between aerobic fitness and metabolic power metrics in elite male soccer players, and the possible differences that playing positions might impose during match play over new metabolic power metrics. Sixty-two elite professional male soccer players (13 central backs, 13 side backs, 22 midfielders, and 14 forwards) took part in the study. Players were monitored during eleven months of full training (including pre-season and in-season) and over all official matches (Serie A matches, Italy Cup matches). Aerobic fitness tests were conducted one week after the start of the preseason, and 8, 24 and 36 weeks after the beginning of the Championship. Players’ aerobic fitness and metabolic power metrics were considered as the mean of all seasonal testing and of pooling data of 38 championship matches and 3 or 6 Italy Cup matches for all the calculations respectively. The velocity at 4 mmol·L^-1 (VL₄) was significantly related to metabolic power metrics match variables with correlation ranging from trivial to very large (r = 0.32 to r = 0.89). Receiver-operating-characteristic (ROC) analysis showed that speed at VL₄ was sensitive in detecting high metabolic power distance (HMPD) changes in all but central back players as revealed by area under the curve (central back .78, 95%CI .47 to .95; full back .93, 95%CI .64 to 0.99; midfielder .88, 95%CI .67 to 0.98; forward .90, 95%CI .62 to 0.99). This study’s findings provide further evidence for the ecological validity of aerobic fitness in elite male soccer players. Players having a HMPD cut-off equal to or higher than > 1450 m for central backs, > 1990 m for full backs, > 2170 m for midfielders and > 1670 m for forwards may be considered as possessing superior aerobic fitness status. In light of this study’s findings, the VL₄ test may be considered a valid test to evaluate meaningful information for direct generic aerobic training in soccer players.     

A Shape-Engineered Surface-Enhanced Raman Scattering Optical Fiber Sensor Working from the Visible to the Near-Infrared

Surface-enhanced Raman scattering (SERS) takes advantage of the giant electromagnetic field enhancement provided by localized surface plasmons in metal nanoparticles to amplify the weak Raman scattering of the molecules. Optical fibers coated with noble metal nanoparticles can therefore be used as SERS-based sensors for remote detection of molecular species. In this article, we report on the development of an optical fiber SERS sensor capable to operate on a range of excitation wavelengths from the visible to the near-infrared. We introduce a quasistatic chemical etching protocol to engineer the tip shape and investigate the effects of the tip shape on the sensor performances.