Changes in serum markers indicative of health effects in vineyard workers following exposure to the fungicide mancozeb: an Italian study

The aim of this study was to investigate the health effects induced by exposure to the fungicide mancozeb in Italian vineyard workers. Ninety-three Italian subjects entered the study - 48 vine-growers intermittently exposed to mancozeb and 45 healthy controls. The subjects were investigated three times: before the seasonal application of pesticides (T0), 30 days after the beginning of the application period (T30), and 45 days after T0 (T45). At T0 the comparison between agricultural workers and controls showed a higher prevalence of cold or flu symptoms, a statistically significant lower percentage of monocytes, higher absolute count of T lymphocytes, CD4 and natural killer cells, and lower plasma levels of IgA and IgM in workers. Such differences were not confirmed at T30 and T45. In fact at T30 in exposed workers, besides a significant increase of urinary ethylenethiourea, confirming mancozeb exposure, T lymphocytes, CD4 and natural killer cells, IgA and IgM returned to values comparable to those observed in controls. Moreover, no other differences in clinical signs, haematological, and immune parameters, such as the immune functional capability evaluated as a response to hepatitis B vaccination, was observed. Altogether the differences between exposed and controls were not consistently correlated to any clinical impairment and suggest that the seasonal application of mancozeb does not pose a significant health risk to exposed subjects.     

Folding, activity and targeting of mutated human cathepsin D that cannot be processed into the double-chain form

The precursor of human cathepsin D (CD) is converted into the single-chain and the double-chain active polypeptides by subsequent proteolysis reactions taking place in the endosomal-lysosomal compartment and involving specific aminoacid sequences. We have mutagenized the region of aminoacids (comprising the beta-hairpin loop) involved in the latter proteolytic maturation step and generated a mutant CD that cannot be converted into the mature double-chain form. This mutant CD expressed in rodent cells reaches the lysosome and is stable as single-chain polypeptide, bears high-mannose type sugars, binds to pepstatin A and is enzymatically active, indicating that it is correctly folded. The present work provides new insights on the aminoacid region involved in the terminal processing of human CD and on the function of the processing beta-hairpin loop.     

Disruption of miR-29 Leads to Aberrant Differentiation of Smooth Muscle Cells Selectively Associated with Distal Lung Vasculature

Differentiation of lung vascular smooth muscle cells (vSMCs) is tightly regulated during development or in response to challenges in a vessel specific manner. Aberrant vSMCs specifically associated with distal pulmonary arteries have been implicated in the pathogenesis of respiratory diseases, such as pulmonary arterial hypertension (PAH), a progressive and fatal disease, with no effective treatment. Therefore, it is highly relevant to understand the underlying mechanisms of lung vSMC differentiation. miRNAs are known to play critical roles in vSMC maturation and function of systemic vessels; however, little is known regarding the role of miRNAs in lung vSMCs. Here, we report that miR-29 family members are the most abundant miRNAs in adult mouse lungs. Moreover, high levels of miR-29 expression are selectively associated with vSMCs of distal vessels in both mouse and human lungs. Furthermore, we have shown that disruption of miR-29 in vivo leads to immature/synthetic vSMC phenotype specifically associated with distal lung vasculature, at least partially due to the derepression of KLF4, components of the PDGF pathway and ECM-related genes associated with synthetic phenotype. Moreover, we found that expression of FBXO32 in vSMCs is significantly upregulated in the distal vasculature of miR-29 null lungs. This indicates a potential important role of miR-29 in smooth muscle cell function by regulating FBXO32 and SMC protein degradation. These results are strongly supported by findings of a cell autonomous role of endogenous miR-29 in promoting SMC differentiation in vitro. Together, our findings suggested a vessel specific role of miR-29 in vSMC differentiation and function by targeting several key negative regulators.     

Lewis acid catalysed methylation of N‐(9H‐fluoren‐9‐yl)methanesulfonyl (Fms) protected lipophilic α‐amino acid methyl esters

This work reports an efficient Lewis acid catalysed N‐methylation procedure of lipophilic α‐amino acid methyl esters in solution phase. The developed methodology involves the use of the reagent system AlCl₃/diazomethane as methylating agent and α‐amino acid methyl esters protected on the amino function with the (9H‐fluoren‐9‐yl)methanesulfonyl (Fms) group. The removal of Fms protecting group is achieved under the same conditions to those used for Fmoc removal. Thus the Fms group can be interchangeable with the Fmoc group in the synthesis of N‐methylated peptides using standard Fmoc‐based strategies. Finally, the absence of racemization during the methylation reaction and the removal of Fms group were demonstrated by synthesising a pair of diastereomeric dipeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

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.