Reproducibility in urine peptidome profiling using MALDI‐TOF

MALDI‐TOF profiling of low molecular weight peptides (peptidome) usage is limited due to the lack of reproducibility from the confounding inferences of sample preparation, data acquisition, and processing. We applied MALDI‐TOF analysis to profile urine peptidome with the aims to: (i) compare centrifugal ultrafiltration and dialysis pretreatments, (ii) determine whether using signal LOD (sLOD), together with data normalization, may reduce MALDI‐TOF variability. We also investigated the influence of peaks detection on reproducibility. Dialysis allowed to obtain better MALDI‐TOF spectra than ultrafiltration. Within the 1000–4000 m/z range, we identified 120 and 129 peaks in intra‐ and interassay studies, respectively. To estimate the sLOD, serial dilution of pooled urines up to 1/256 were analyzed in triplicate. Six data normalization strategies were investigated–the mean, median, internal standard, relative intensity, TIC, and linear rescaling normalization. Normalization methods alone performed poorly in reducing features variability while when combined to sLOD adjustment showed an overall reduction in features CVs. Applying a feedback signal processing approach, after median normalization and sLOD adjustment, CVs were reduced from 103 to 26% and 113 to 25% for the intra‐ and interassay, respectively, and spectra became more comparable in terms of data dispersion.     

Peroxisomal fatty acid uptake mechanism in Saccharomyces cerevisiae

Peroxisomes play a major role in human cellular lipid metabolism, including fatty acid β-oxidation. The most frequent peroxisomal disorder is X-linked adrenoleukodystrophy, which is caused by mutations in ABCD1. The biochemical hallmark of X-linked adrenoleukodystrophy is the accumulation of very long chain fatty acids (VLCFAs) due to impaired peroxisomal β-oxidation. Although this suggests a role of ABCD1 in VLCFA import into peroxisomes, no direct experimental evidence is available to substantiate this. To unravel the mechanism of peroxisomal VLCFA transport, we use Saccharomyces cerevisiae as a model organism. Here we provide evidence that in this organism very long chain acyl-CoA esters are hydrolyzed by the Pxa1p-Pxa2p complex prior to the actual transport of their fatty acid moiety into the peroxisomes with the CoA presumably being released into the cytoplasm. The Pxa1p-Pxa2p complex functionally interacts with the acyl-CoA synthetases Faa2p and/or Fat1p on the inner surface of the peroxisomal membrane for subsequent re-esterification of the VLCFAs. Importantly, the Pxa1p-Pxa2p complex shares this molecular mechanism with HsABCD1 and HsABCD2.     

Inter-electrode spacing of surface EMG sensors: reduction of crosstalk contamination during voluntary contractions

We investigated the influence of inter-electrode spacing on the degree of crosstalk contamination in surface electromyographic (sEMG) signals in the tibialis anterior (target muscle), generated by the triceps surae (crosstalk muscle), using bar and disk electrode arrays. The degree of crosstalk contamination was assessed for voluntary constant-force isometric contractions and for dynamic contractions during walking. Single-differential signals were acquired with inter-electrode spacing ranging from 5 mm to 40 mm. Additionally, double differential signals were acquired at 10 mm spacing using the bar electrode array. Crosstalk contamination at the target muscle was expressed as the ratio of the detected crosstalk signal to that of the target muscle signal. The crosstalk contamination ratio approached a mean of 50% for the 40 mm spacing for triceps surae muscle contractions at 80% MVC and tibialis anterior muscle contractions at 10% MVC. For single differential recordings, the minimum crosstalk contamination was obtained from the 10 mm spacing. The results showed no significant differences between the bar and disk electrode arrays. During walking, the crosstalk contamination on the tibialis anterior muscle reached levels of 23% for a commonly used 22 mm spacing single-differential disk sensor, 17% for a 10 mm spacing single-differential bar sensor, and 8% for a 10 mm double-differential bar sensor. For both studies the effect of electrode spacing on crosstalk contamination was statistically significant. Crosstalk contamination and inter-electrode spacing should therefore be a serious concern in gait studies when the sEMG signal is collected with single differential sensors. The contamination can distort the target muscle signal and mislead the interpretation of its activation timing and force magnitude.     

Differential susceptibility in a specialised aphidophagous ladybird,Platynaspis luteorubra (Coleoptera: Coccinellidae), facing intraguild predation by exotic and native generalist predators

The establishment of exotic generalist predators raises concern about possible domination of the invaded community and displacement of native species. In this respect, recent studies suggest that specialists are likely to be more at risk of niche displacement than generalists, thereby compromising biological control in agroecosystems. Under laboratory conditions, we examined the susceptibility of a specialised myrmecophilous ladybird, Platynaspis luteorubra Goeze, as intraguild (IG) prey of generalist native (Oenopia conglobata L. and Adalia bipunctata L.) and exotic (Harmonia axyridis Pallas) coccinellid species. We described parameters of predatory behaviour (for example, attack rate and effectiveness of antipredator defence) and their variation among predators. Furthermore, we estimated the effect of these parameters on the time to predation using a Cox proportional hazards model. We found that A. bipunctata was the most effective IG predator of P. luteorubra, with the highest rate of successful attacks; however, P. luteorubra was not very susceptibile to attack by O. conglobata or H. axyridis, and these species exhibited lower rates of successful attack and predation. Our results, together with field observations, suggest that IG predation is a factor that fosters niche specialization in P. luteorubra and decreases the realised niche of this species in central Italy. Furthermore the lower susceptibility of P. luteorubra to H. axyridis may be due to a lack of historical co-occurrence between preimmaginal stages, possibly arising from differental selection of oviposition sites.     

Selected lactic acid bacteria synthesize antioxidant peptides during sourdough fermentation of cereal flours

A pool of selected lactic acid bacteria was used for the sourdough fermentation of various cereal flours with the aim of synthesizing antioxidant peptides. The radical-scavenging activity of water/salt-soluble extracts (WSE) from sourdoughs was significantly (P < 0.05) higher than that of chemically acidified doughs. The highest activity was found for whole wheat, spelt, rye, and kamut sourdoughs. Almost the same results were found for the inhibition of linoleic acid autoxidation. WSE were subjected to reverse-phase fast protein liquid chromatography. Thirty-seven fractions were collected and assayed in vitro. The most active fractions were resistant to further hydrolysis by digestive enzymes. Twenty-five peptides of 8 to 57 amino acid residues were identified by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry. Almost all of the sequences shared compositional features which are typical of antioxidant peptides. All of the purified fractions showed ex vivo antioxidant activity on mouse fibroblasts artificially subjected to oxidative stress. This study demonstrates the capacity of sourdough lactic acid bacteria to release peptides with antioxidant activity through the proteolysis of native cereal proteins.     

The phosphodiesterases type 5 inhibitor tadalafil reduces the activation of the hypothalamus-pituitary-adrenal axis in men during cycle ergometric exercise

Phosphodiesterase type 5 inhibitors may influence human physiology, health, and performance by also modulating endocrine pathways. We evaluated the effects of a 2-day tadalafil administration on adenohypophyseal and adrenal hormone adaptation to exercise in humans. Fourteen healthy males were included in a double-blind crossover trial. Each volunteer randomly received two tablets of placebo or tadalafil (20 mg/day with a 36-h interval) before a maximal exercise was performed. After a 2-wk washout, the volunteers were crossed over. Blood samples were collected at -30 and -15 min and immediately before exercise, immediately after, and during recovery (+15, +30, +60, and +90 min) for adrenocorticotropin (ACTH), β-endorphin, growth hormone (GH), prolactin, cortisol (C), corticosterone, dehydroepiandrosterone-sulfate (DHEAS), and cortisol binding globulin (CBG) assays. C-to-CBG (free cortisol index, FCI) and DHEAS-to-C ratios were calculated. Exercise intensity, perceived exertion rate, O? consumption, and CO? and blood lactate concentration were evaluated. ACTH, GH, C, corticosterone, and CBG absolute concentrations and/or areas under the curve (AUC) increased after exercise after both placebo and tadalafil. Exercise increased DHEAS only after placebo. Compared with placebo, tadalafil administration reduced the ACTH, C, corticosterone, and FCI responses to exercise and was associated with higher β-endorphin AUC and DHEAS-to-C ratio during recovery, without influencing cardiorespiratory and performance parameters. Tadalafil reduced the activation of the hypothalamus-pituitary-adrenal axis during exercise by probably influencing the brain's nitric oxide- and cGMP-mediated pathways. Further studies are necessary to confirm our results and to identify the involved mechanisms, possible health risks, and potential clinical uses.     

Conformational Flexibility and Absolute Stereochemistry of (3R)-3-hydroxy-4-aryl-β-lactams Investigated by Chiroptical Properties and TD-DFT Calculations

The effect of conformational flexibility on the chiroptical properties of a series of synthetic (3R)-3-hydroxy-4-aryl-β-lactams of known stereochemistry (1-6) was investigated by means of electronic circular dichroism (ECD) measurements and time-dependent density functional theory (TD-DFT) calculations. The application of the β-lactam sector rules allowed a correct stereochemical characterization of these compounds, with the exception of a thienyl-substituted derivative (cis-). TD-DFT calculations yielded accurate predictions of experimental ECD spectra and [α](D) values, allowing us to assign the correct absolute configuration to all the investigated compounds. A detailed analysis of the β-lactam ring equilibrium geometry on optimized conformers identified regular patterns for the arrangement of atoms around the amide chromophore, confirming the validity of the β-lactam sector rules. However, relevant variations in theoretical chiroptical properties were found for compounds bearing a heterocyclic substituent at C4 or a phenyl substituent at C3, whose conformers deviate from these regular geometric patterns. This behavior explains the failure of the β-lactam sector rules in cis-. This study showed the importance of conformational flexibility for the determination of chiroptical properties and highlighted the strengths and weaknesses of the different methods for the stereochemical characterization of chiral molecules in solution. Chirality 24:741-750, 2012. ? 2012 Wiley Periodicals, Inc.     

Dakin–West reaction on 1-thyminyl acetic acid for the synthesis of 1,3-bis(1-thyminyl)-2-propanone, a heteroaromatic compound with nucleopeptide-binding properties

This work deals with the Dakin-West synthesis, starting from the nucleoamino acid 1-thyminyl acetic acid, as well the NMR, ESI MS, and X-ray characterization of a heteroaromatic compound denominated by us T(2)CO, comprising two thymine moieties anchored to a 2-propanonic unit, the spectroscopic properties of which were studied by UV as a function of temperature and ionic strength. Preliminary binding-studies with molecules of biomedical interest such as nucleic acids and proteins, performed on samples containing T(2)CO, suggested that this molecule is able to interact very weakly with double-stranded RNA, whereas it does not seem to bind other nucleic acids or proteins. Moreover, by studies with fresh human serum we found that T(2)CO is resistant to enzymatic degradation till 24?h, whereas UV metal binding-studies, performed using solutions of copper (II) chloride dihydrate and nickel (II) chloride hexahydrate, revealed a certain ability of T(2)CO to bind copper (II) cation. Finally, by CD spectroscopy we investigated the influence of T(2)CO on the already described supramolecular networks based on L-serine-containing nucleopeptides. More particularly, we found that T(2)CO is able to increase the level of structuration of the non-covalent supramolecular assembly of the chiral nucleopeptides, which is a feature of remarkable interest for the development of innovative drug delivery tools.