Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases

Phytocystatins are inhibitors of cysteine-proteases from plants putatively involved in plant defence based on their capability of inhibit heterologous enzymes. We have previously characterised the whole cystatin gene family members from barley (HvCPI-1 to HvCPI-13). The aim of this study was to assess the effects of barley cystatins on two phytophagous spider mites, Tetranychus urticae and Brevipalpus chilensis. The determination of proteolytic activity profile in both mite species showed the presence of the cysteine-proteases, putative targets of cystatins, among other enzymatic activities. All barley cystatins, except HvCPI-1 and HvCPI-7, inhibited in vitro mite cathepsin L- and/or cathepsin B-like activities, HvCPI-6 being the strongest inhibitor for both mite species. Transgenic maize plants expressing HvCPI-6 protein were generated and the functional integrity of the cystatin transgene was confirmed by in vitro inhibitory effect observed against T. urticae and B. chilensis protein extracts. Feeding experiments impaired on transgenic lines performed with T. urticae impaired mite development and reproductive performance. Besides, a significant reduction of cathepsin L-like and/or cathepsin B-like activities was observed when the spider mite fed on maize plants expressing HvCPI-6 cystatin. These findings reveal the potential of barley cystatins as acaricide proteins to protect plants against two important mite pests

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Identification of Yeasts Is Contingent on Robust Reference Spectra

BACKGROUND: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for yeast identification is limited by the requirement for protein extraction and for robust reference spectra across yeast species in databases. We evaluated its ability to identify a range of yeasts in comparison with phenotypic methods. METHODS: MALDI-TOF MS was performed on 30 reference and 167 clinical isolates followed by prospective examination of 67 clinical strains in parallel with biochemical testing (total n = 264). Discordant/unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene cluster. PRINCIPAL FINDINGS: Twenty (67%; 16 species), and 24 (80%) of 30 reference strains were identified to species, (spectral score ≥2.0) and genus (score ≥1.70)-level, respectively. Of clinical isolates, 140/167 (84%) strains were correctly identified with scores of ≥2.0 and 160/167 (96%) with scores of ≥1.70; amongst Candida spp. (n = 148), correct species assignment at scores of ≥2.0, and ≥1.70 was obtained for 86% and 96% isolates, respectively (vs. 76.4% by biochemical methods). Prospectively, species-level identification was achieved for 79% of isolates, whilst 91% and 94% of strains yielded scores of ≥1.90 and ≥1.70, respectively (100% isolates identified by biochemical methods). All test scores of 1.70-1.90 provided correct species assignment despite being identified to "genus-level". MALDI-TOF MS identified uncommon Candida spp., differentiated Candida parapsilosis from C. orthopsilosis and C. metapsilosis and distinguished between C. glabrata, C. nivariensis and C. bracarensis. Yeasts with scores of <1.70 were rare species such as C. nivariensis (3/10 strains) and C. bracarensis (n = 1) but included 4/12 Cryptococcus neoformans. There were no misidentifications. Four novel species-specific spectra were obtained. Protein extraction was essential for reliable results. CONCLUSIONS: MALDI-TOF MS enabled rapid, reliable identification of clinically-important yeasts. The addition of spectra to databases and reduction in identification scores required for species-level identification may improve its utility

Dioxins in Food and Feed - Reference Methods and New Certified Reference Materials (DIFFERENCE)

The European research project DIFFERENCE (“Dioxins in Food and Feed – Reference methods and New Certified Reference Materials”) was focussed on the development of an alternative methods for analysis of polychlorinated dibenzodioxins (PCDDs), dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dl-PCBs) using comprehensive multi-dimensional gas chromatography (GC×GC), gas chromatography combined with low resolution ion-trap mass spectrometry (GCLRMS/ MS), the CALUX bioassay and an Ah-PCR technique. GC combined with high resolution mass spectrometry (HRMS) was used as a reference method in all comparisons

Cognitive task avoidance correlates with fatigue-induced performance decrement but not with subjective fatigue

Mentally demanding tasks feel effortful and are usually avoided. Furthermore, prolonged cognitive engagement leads to mental fatigue, consisting of subjective feeling of exhaustion and decline in performance. Despite the intuitive characterization of fatigue as an increase in subjective effort perception, the effect of fatigue on effort cost has never been tested experimentally. To this end, sixty participants in 2 separate experiments underwent a forced-choice working memory task following either a fatigue-inducing (i.e. cognitive task involving working memory, conflict and switch costs) or a control manipulation. We measured fatigue in terms of subjective feeling and performance decrement and assessed effort in terms of subjective perception and task avoidance. Subjects exhibited only weak avoidance of the working memory task, with stronger influence of reward than task difficulty on their decisions. In addition, we found that task avoidance did not systematically change following the fatigue manipulation but that variations in task avoidance correlated with fatigue-induced performance decline. The other measures of fatigue and effort were unrelated to each other. Our findings suggest that subjective fatigue may develop independently of task avoidance and suggest an "anticipatory regulation" model in which fatigue urges subjects to stop in anticipation of possible, future adverse consequences

Tracking the sterol biosynthesis pathway of the diatom <i>Phaeodactylum tricornutum</i>

Diatoms are unicellular photosynthetic microalgae that play a major role in global primary production and aquatic biogeochemical cycling. Endosymbiotic events and recurrent gene transfers uniquely shaped the genome of diatoms, which contains features from several domains of life. The biosynthesis pathways of sterols, essential compounds in all eukaryotic cells, and many of the enzymes involved are evolutionarily conserved in eukaryotes. Although well characterized in most eukaryotes, the pathway leading to sterol biosynthesis in diatoms has remained hitherto unidentified.Through the DiatomCyc database we reconstructed the mevalonate and sterol biosynthetic pathways of the model diatom Phaeodactylum tricornutum in silico. We experimentally verified the predicted pathways using enzyme inhibitor, gene silencing and heterologous gene expression approaches.Our analysis revealed a peculiar, chimeric organization of the diatom sterol biosynthesis pathway, which possesses features of both plant and fungal pathways. Strikingly, it lacks a conventional squalene epoxidase and utilizes an extended oxidosqualene cyclase and a multifunctional isopentenyl diphosphate isomerase/squalene synthase enzyme.The reconstruction of the P. tricornutum sterol pathway underscores the metabolic plasticity of diatoms and offers important insights for the engineering of diatoms for sustainable production of biofuels and high-value chemicals.</li

Dioxins in Food and Feed - Reference Methods and New Certified Reference Materials (DIFFERENCE)

The European research project DIFFERENCE (“Dioxins in Food and Feed – Reference methods and New Certified Reference Materials”) was focussed on the development of an alternative methods for analysis of polychlorinated dibenzodioxins (PCDDs), dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dl-PCBs) using comprehensive multi-dimensional gas chromatography (GC×GC), gas chromatography combined with low resolution ion-trap mass spectrometry (GCLRMS/ MS), the CALUX bioassay and an Ah-PCR technique. GC combined with high resolution mass spectrometry (HRMS) was used as a reference method in all comparisons