DypB peroxidase for aflatoxin removal: New insights into the toxin degradation process.

Aflatoxin B1 (AFB1) is one of the most potent carcinogens and a widespread food and feed contaminant. As for other toxins, many efforts are devoted to find efficient and environmentally-friendly methods to degrade AFB1, such as enzymatic treatments, thus improving the safety of food and feed products. In this regard, the dye decolorizing peroxidase of type B (DypB) can efficiently degrade AFB1. The molecular mechanism, which is required to drive protein optimization in view of the usage of DypB as a mycotoxin reduction agent in large scale application, is unknown. Here, we focused on the role of four DypB residues in the degradation of AFB1 by alanine-scanning (residues 156, 215, 239 and 246), which were identified from biochemical assays to be kinetically relevant for the degradation. As a result of DypB degradation, AFB1 is converted into four products. Interestingly, the relative abundancy of these products depends on the replaced residues. Molecular dynamics simulations were used to investigate the role of these residues in the binding step between protein and manganese, a metal ion which is expected to be involved in the degradation process. We found that the size of the haem pocket as well as conformational changes in the protein structure could play a role in determining the kinetics of AFB1 removal and, consequently, guide the process towards specific degradation products.

Evaluation of the prevalence of dental agenesis through the use of orthopantomography in a sample of subjects residing in Lombardy and Piedmont regions.

AIM: Dental agenesis is one of the most frequent dental anomalies, with a prevalence varying from 1.6% to 36.5%, depending on the populations studied. The patient's age at diagnosis, sex, and ethnic differences are considered possible influenting factors that can explain such a wide range of prevalence. The objective of the study was to define the frequency of dental agenesis in a sample of subjects living in Piedmont and Lombardy regions of Italy. MATERIALS: X-rays, already taken for other diagnostic purposes, were collected. Orthopantomographies belonging to subjects born after 1995 and aged between 7.9 and 16.9 years were selected. It was assessed the presence of each tooth, except for third molars since they are frequently absent due to their variability. If a tooth was missing and the patient had additional radiographs, the other radiographs were evaluated to confirm the diagnosis or to rule out a delayed calcification or the presence of a malposition tooth. Results: Orthopantomographies were collected from 1,020 subjects and 98 of them presented agenesis, with a prevalence of 5% for females and 4.61% for males. The most affected teeth were 35 and 45, followed by 12 and 22. The lower arch was more frequently involved by agenesis: there were 107 teeth absent in the mandibular arch and 83 in the maxillary arch.

Antimicrobial resistance gene shuffling and a three-element mobilisation system in the monophasic Salmonella typhimurium strain ST1030.

In this study we describe the genetic elements and the antimicrobial resistance units (RUs) harboured by the Salmonella Typhimurium monophasic variant 1,4,[5],12:i:- strain ST1030. Of the three identified RUs two were chromosomal, RU1 (IS26-blaTEM-1-IS26-strAB-sul2- IS26) and RU2 (IS26-tetR(B)-tetA(B)-ΔIS26), and one, RU3 (a sul3-associated class 1 integron with cassette array dfrA12-orfF-aadA2-cmlA1-aadA1), was embedded in a Tn21-derived element harboured by the conjugative I1 plasmid pST1030-1A. IS26 elements mediated the antimicrobial resistance gene (ARG) shuffling and this gave rise to pST1030-1A derivatives with different sets of ARGs. ST1030 also harboured two ColE1-like plasmids of which one, pST1030-2A, was mobilisable and the target of an intracellular translocation of the Tn21-derived element; the second (pST1030-3) was an orphan mob-associated oriT plasmid co-transferred with pST1030-1A and pST1030-2A. pST1030-2A and pST1030-3 also carried a parA gene and a type III restriction modification system, respectively. Overall analysis of our data reinforces the role played by IS26, Tn21-derived elements and non-conjugative plasmids in the spread of ARGs and supplies the first evidence, at least in Salmonella, for the identification of a natural isolate harbouring a three-element mobilisation system in the same cell.

Application of a recombinant laccase-chlorogenic acid system in protein crosslink and antioxidant properties of the curd.

Milk protein crosslink through the action of enzymes represents a feasible strategy to impart new functionalities to cheese. In this work we reported the effects of a laccase mediator system (LMS) on protein crosslink and antioxidant property of curd. The crosslinking activity of a purified recombinant laccase Ery4 and a commercial enzyme preparation (cLC), with three mediators was firstly evaluated in milk and then applied before curd manufacture. Only Ery4-LMS significantly increased curd weight compared to that of the control sample. SDS-PAGE revealed that similar high molecular weight bands produced by both LMSs in milk were also retained in curds. The antioxidant activity recorded in curds with Ery4-LMS was the highest among all samples both before and after gastro-pancreatic digestion. This is the first time that a CGA-based LMS is used in manufacture of curd with improved antioxidant properties. These results open new perspectives for dairy applications.

Serum uric acid is not independently associated with plasma renin activity and plasma aldosterone in hypertensive adults.

BACKGROUND AND AIMS: In experimental investigations conducted in rats, raising serum uric acid (SUA) levels resulted in the stimulation of intrarenal renin expression. Studies in humans exploring the association of SUA with plasma renin activity (PRA) yielded conflicting results. Moreover, little is known about the relationship of SUA with plasma aldosterone concentration (PAC). The study aimed to assess the relationship between SUA levels, PRA, and PAC and the influence of age, gender, body mass index (BMI), and hyperuricemia on these relationships in subjects with essential hypertension (EH). METHODS AND RESULTS: We enrolled 372 hypertensive patients (mean age 45 ± 12 years, men 67%) with uncomplicated EH that was not pharmacologically treated. The study population was divided in tertiles according to SUA levels. While PRA did not differ significantly across the three tertiles, PAC was higher in subjects belonging to the uppermost tertile of SUA than those in the lower ones (p = 0.0429); however, this difference lost statistical significance after adjustment for age, sex, BMI, and serum creatinine. Univariate correlation analyses showed significant associations of SUA with PRA (r = 0.137; p = 0.008) and PAC (r = 0.179; p < 0.001). However, these relationships were not significant after correcting for confounding factors in multiple linear regression analyses. We did not observe statistically significant effect modification by gender, age, BMI, and hyperuricemia. CONCLUSION: SUA levels are weakly associated with PRA and PAC in adults with untreated EH. These relationships were lost after adjustment for age, sex, BMI, and serum creatinine.

Average real variability of 24-h systolic blood pressure is associated with microalbuminuria in patients with primary hypertension.

Limited and conflicting data are available about the association between short-term blood pressure (BP) variability and urinary albumin excretion rate (uAER). The objective of our study was to analyze the relationships between microalbuminuria (MAU), defined as an uAER between 20 and 200 µg min(-1), and short-term BP variability (BPV), assessed as average real variability (ARV), weighted s.d. of 24-h BP and as s.d. of daytime and night-time BP. The study population consisted of 315 untreated essential hypertensives with normal estimated glomerular filtration rate (>60 ml min(-1) per 1.73 m(2)), who underwent 24-h ambulatory BP monitoring and 24-h uAER determination. MAU was detected in 82 (26%) patients. ARV of 24-h systolic BP (SBP) was significantly higher in patients with MAU (9.8 (8.5-11.1) mm Hg) when compared with those without it (9.1 (8-10.2) mm Hg; P=0.007). This difference held (P=0.026) after adjustment for age, mean levels of BP and other potential confounders by analysis of covariance. A statistically significant correlation was also found between ARV of 24-h SBP and uAER (r=0.17; P=0.003). This association remained significant (ß=0.15; P=0.01), also taking into account the effect of 24-h average systolic and diastolic BP, age, gender, diabetes, serum uric acid, triglycerides, estimated glomerular filtration rate in multiple regression analyses. All the other indices of short-term BPV tested were not independently associated with MAU. Our results seem to suggest that in essential hypertension, short-term BPV, only when estimated by ARV of 24-h SBP, is independently associated with MAU.

Renal haemodynamics and severity of carotid atherosclerosis in hypertensive patients with and without impaired renal function.

BACKGROUND AND AIM: Renal resistance index (RRI), assessed by Duplex-Doppler sonography, has been classically considered as a mere expression of intrarenal vascular resistance. Recent studies, however, have showed that RRI is also influenced by upstream factors, especially arterial compliance, confirming its possible role as a marker of systemic vascular alterations. Several studies have shown that carotid intima-media thickness (cIMT) and carotid plaques (cP), assessed by ultrasonography, are documented markers of subclinical organ damage as well as expression of progressive atherosclerotic disease, and that they get worse with the progressive deterioration of renal function. The study was aimed to evaluate the relationship between RRI and severity of carotid atherosclerosis in hypertensive subjects with and without impaired renal function. METHODS AND RESULTS: The study population, including 263 hypertensive patients (30-70 years), was split into 3 groups based on cIMT and presence of cP (cIMT ≤ 0.9 mm and no cP; cIMT > 0.9 mm and no cP; cP). All patients were also divided into 2 subgroups (normal renal function; CKD stage I-IV). A stepwise increase in RRI corresponding to the groups of progressive severity of carotid atherosclerosis was observed (respectively 0.61 ± 0.07, 0.65 ± 0.06, 0.68 ± 0.06; p < 0.001). A strong positive correlation was observed between RRI and cIMT in the whole population (r = 0.43; p < 0.001) and in the subgroups with (r = 0.42; p < 0.001) and without (r = 0.39; p < 0.001) CKD. These associations remained statistically significant even after adjustment for various confounding factors. CONCLUSION: Showing a close association between RRI and severity of carotid atherosclerosis, our results strengthen the concept that RRI is a marker of systemic vascular changes.

Vitamin D receptor gene polymorphisms and plasma renin activity in essential hypertensive individuals.

Several studies analyzed 25-hydroxyvitamin D (25[OH]D) and blood pressure (BP) relationship with mixed results. Moreover, a relationship between the risk of hypertension and vitamin D receptor (VDR) gene polymorphisms, FokI and BsmI, was reported. This study was aimed to analyze these relationships in essential hypertensive (EH) patients. Seventy-one EH patients, 18-75 years old, were enrolled. Patients underwent clinical BP, 24-h ambulatory BP monitoring, 25[OH]D and plasma renin activity (PRA) evaluations. FokI and BsmI VDR polymorphisms were analyzed and compared with those of 72 healthy controls. In EH patients, the median 25[OH]D levels were lower than 30 ng ml(-1). We found a significant negative correlation between 25[OH]D and 24-h systolic BP (r = -0.277, P = 0.043). This correlation persisted in backward stepwise multivariate analyses (ß = -0.337; P = 0.022), after adjustment for age, gender, body mass index, glomerular filtration rate, and PRA. We did not observe statistically significant correlation between 25[OH]D and PRA. We compared the allelic frequencies and genotype distribution between patients and controls, and FokI and BsmI VDR polymorphisms were not associated either with hypertensive status or with PRA. Further wide studies are needed to clarify this relationship.

Relationships between mild hyperuricaemia and aortic stiffness in untreated hypertensive patients.

BACKGROUND AND AIMS: Clinical studies exploring the relationship between serum uric acid (SUA) and arterial stiffness yielded conflicting results. Only in a few of these studies, arterial distensibility was examined by measuring aortic pulse wave velocity (PWV), which is considered the gold standard for evaluating arterial stiffness. In none of the previous investigations was the influence of SUA on aortic distensibility assessed, taking into account the effect of albuminuria. The purpose of our study was to comprehensively analyse the relationships between SUA and aortic PWV in a group of essential hypertensive patients. METHODS AND RESULTS: We enrolled 222 untreated and uncomplicated hypertensive subjects (mean age: 44 ± 10 years; 60% males), without gout. In all patients, SUA and urinary albumin excretion rate (AER) were determined. Moreover, carotid-femoral (c-f) PWV was measured. C-f PWV was significantly higher in hypertensive patients belonging to the uppermost tertile of SUA distribution, compared to subjects of the lowest tertiles (10.9 ± 2.2 vs. 10 ± 1.8 vs. 9.9 ± 1.7 m s(-1); p = 0.001). In univariate analysis, SUA correlated with c-f PWV (r = 0.24; p < 0.001). This association disappeared when AER was added in a multiple regression model, including SUA, age, mean arterial pressure, gender, metabolic syndrome components and glomerular filtration rate. CONCLUSION: The results of our study showed that, in essential hypertensive subjects, there is a positive relationship between mild hyperuricaemia and aortic stiffness. This association weakened after adjustment for covariates and lost statistical significance after further correction for albuminuria.

Aspergillus section Nigri as contributor of fumonisin B(2) contamination in maize.

Fumonisins (FBs), which are carcinogenic mycotoxins, are known to be typically produced by several phytopathogenic fungal species belonging to the genus Fusarium. F. proliferatum and F. verticillioides, two important pathogens of maize worldwide, are the most common species that produce FBs. The main FBs produced by these species are FB1, FB2 and FB3. Moreover, recently, fungal strains belonging to Aspergillus niger have been also reported to produce FBs (in particular, FB2 and FB4). In a survey on maize carried out in Central Italy, 17 maize kernel samples were collected at harvest and analysed for FB1, FB2 and FB3, as well as fungal contamination, with a particular attention to the species-producing FBs. All 17 samples were contaminated by F. verticillioides and/or F. proliferatum at a level ranging from 13% to 100% of kernels. However, 10 out of 17 samples were also contaminated by Aspergillus section Nigri with a range from 6% to 68% of kernels. There was a significant inverse logarithmic relationship between levels of Fusarium and Aspergillus contamination. All samples were contaminated by FBs; FB1 ranged from 0.09 to 30.2 µg g(-1), whereas FB2 ranged from 0.04 to 13.2 µg g(-1). The ratio of FB2/FB1 contamination in the maize samples was evaluated and the highest values occurred in samples contaminated with Aspergillus section Nigri. Thirty strains of Aspergillus section Nigri isolated from these samples were molecularly identified (based on sequences of two housekeeping genes) and analysed for their capability to produce FB2. Among the 30 strains isolated, 12 were identified as Aspergillus welwitschiae (syn. A. awamori) and 18 as A. tubingensis. FB2 was produced by five out of 12 strains of A. welwitschiae within a range of 0.20-5 µg g(-1). This is the first report showing the capability of Aspergillus section Nigri from maize to produce FB2 and its possibility to contribute to FB accumulation in kernels.

Transcriptional regulation of enniatins production by Fusarium avenaceum.

AIMS: The aim of this study was to analyse the transcriptional regulation of enniatins (ENs) production in Fusarium avenaceum. METHODS AND RESULTS: We develop a new method to quantify ENs in FDM agar medium. We performed an LC/MS/MS analysis to evaluate enniatin A, A1, B, B1 and B4 production by seven F. avenaceum strains and, in a time-course experiment, by ITEM 3404 to analyse the transcriptional regulation of the esyn1 gene. The expression profile, achieved by Real time reverse transcriptase assay, showed an activation of gene transcription at the seventh day of incubation, corresponding to the higher increase of total ENs production. Enniatin B was the most abundant ENs analogues, representing the 90% of total ENs. The relative percentage of ENs remained unaltered during the experiment. CONCLUSIONS: We reported a transcriptional regulation of esyn1 responsible for the modulation of ENs biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Enniatins are cyclic depsipeptides metabolites with a wide range of biological activities. They are also widespread contaminants in grains and cereals due to infection by enniatin-producing Fusarium species. This is the first article describing the transcriptional regulation of esyn1 gene that modulates ENs production in Fusarium avenaceum and provides new knowledge about the molecular mechanism underlying the biosynthesis of these important fungal metabolites in this toxigenic fungal species.

Molecular biodiversity of mycotoxigenic fungi that threaten food safety.

Fungal biodiversity is one of the most important contributors to the occurrence and severity of mycotoxin contamination of crop plants. Phenotypic and metabolic plasticity has enabled mycotoxigenic fungi to colonize a broad range of agriculturally important crops and to adapt to a range of environmental conditions. New mycotoxin-commodity combinations provide evidence for the ability of fungi to adapt to changing conditions and the emergence of genotypes that confer enhanced aggressiveness toward plants and/or altered mycotoxin production profiles. Perhaps the most important contributor to qualitative differences in mycotoxin production among fungi is variation in mycotoxin biosynthetic genes. Molecular genetic and biochemical analyses of toxigenic fungi have elucidated specific differences in biosynthetic genes that are responsible for intra- and inter-specific differences in mycotoxin production. For Aspergillus and Fusarium, the mycotoxigenic genera of greatest concern, variation in biosynthetic genes responsible for production of individual families of mycotoxins appears to be the result of evolutionary adaptation. Examples of such variation have been reported for: a) aflatoxin biosynthetic genes in Aspergillus flavus and Aspergillus parasiticus; b) trichothecene biosynthetic genes within and among Fusarium species; and c) fumonisin biosynthetic genes in Aspergillus and Fusarium species. Understanding the variation in these biosynthetic genes and the basis for variation in mycotoxin production is important for accurate assessment of the risks that fungi pose to food safety and for prevention of mycotoxin contamination of crops in the field and in storage.

Absence of an independent association between serum uric acid and left ventricular mass in Caucasian hypertensive women and men.

BACKGROUND AND AIM: Experimentally uric acid may induce cardiomyocyte growth and interstitial fibrosis of the heart. However, clinical studies exploring the relationship between serum uric acid (SUA) and left ventricular (LV) mass yielded conflicting results. The aim of our study was to evaluate the relationships between SUA and LV mass in a large group of Caucasian essential hypertensive subjects. METHODS AND RESULTS: We enrolled 534 hypertensive patients free of cardiovascular complications and without severe renal insufficiency. In all subjects routine blood chemistry, including SUA determination, echocardiographic examination and 24 h ambulatory blood pressure (BP) monitoring were obtained. In the overall population we observed no significant correlation of SUA with LV mass indexed for height(2.7) (LVMH(2.7)) (r = 0.074). When the same relationship was analysed separately in men and women, we found a statistically significant correlation in female gender (r = 0.27; p < 0.001), but not in males (r = -0.042; p = NS). When we grouped the study population in sex-specific tertiles of SUA, an increase in LVMH(2.7) was observed in the highest tertiles in women (44.5 ± 15.6 vs 47.5 ± 16 vs 55.9 ± 22.2 g/m(2.7); p < 0.001), but not in men. The association between SUA and LVMH(2.7) in women lost statistical significance in multiple regression analyses, after adjustment for age, 24 h systolic BP, body mass index, serum creatinine and other potential confounders. CONCLUSIONS: Our findings do not support an independent association between SUA and LV mass in Caucasian men and women with arterial hypertension.

Young woman with branchio-oto-renal syndrome and a novel mutation in the EYA-1 gene.

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disease clinically characterized by the coexistence of some or all of the following major disorders: deafness, cervical branchial fistulae, preauricular pits, and renal abnormalities. Most families with BOR syndrome have mutations on the EYA-1 gene on chromosome 8q. We present the case of a 23-year-old Italian woman without a familial history of BOR syndrome. The patient, who had hearing loss and a history of surgeries for correction of bilateral cervical branchial fistulae and bilateral preauricular pits, presented with renal impairment, hypertension and overt proteinuria. DNA sequencing showed a novel heterozygous mutation 1420-1421delCC in exon 14 of EYA-1 gene.

Phylogenetic characterization and ochratoxin A--fumonisin profile of black Aspergillus isolated from grapes in Argentina.

Aspergillus section Nigri populations isolated from seven growing regions from Argentina were characterized by sequencing in order to identify species responsible for production of ochratoxin A (OTA) and fumonisins (FB(s)). Sequences of genes encoding calmodulin, ß-tubulin, the second largest subunit of RNA polymerase II and translation elongation factor 1 alpha were analysed. The phylogenetic analysis showed the presence of six lineages: A. carbonarius, A. tubingensis, A. niger, A. japonicus, A. homomorphus and A. foetidus grouped in four major clusters. The molecular tools used allowed the identification for the first time of A. homomorphus from vineyards. OTA production confirmed the importance of A. carbonarius as the main ochratoxigenic species isolated and, to a variable degree, of A. niger and A. tubingensis, which were by far the most commonly occurring species on grapes in Argentina. The only strains able to produce OTA and fumonisins (B(2)-B(4)) belong to the A. niger cluster.

Impact of type 2 diabetes on left ventricular geometry and diastolic function in hypertensive patients with chronic kidney disease.

Left ventricular hypertrophy (LVH) and diastolic dysfunction are very common in patients with chronic kidney disease (CKD). Aim of this study was to evaluate the impact of type 2 diabetes on LV geometry and diastolic function in hypertensive patients with CKD. We enrolled 288 Caucasian subjects with hypertension and CKD; of them, 112 had diabetes. Patients with cardiovascular (CV) diseases, glomerular filtration rate (GFR) >60 ml min(-1) per 1.73 m(2), dialysis treatment and other major non-CV diseases were excluded. All patients underwent routine biochemical analyses and echocardiographic examination with tissue Doppler imaging (TDI). Patients with diabetes had significantly higher LV wall thicknesses (P=0.0001), relative wall thickness (RWT) (P=0.0001) and left atrium volume index (P=0.03), when compared with patients without diabetes. Further, diabetic patients had very high prevalence of concentric LVH. Em, evaluated by TDI, was significantly lower in patients with diabetes (P=0.005). However, the difference lost statistical significance after correction by analysis of covariance for RWT. Multiple stepwise linear regression analysis showed that the variables independently associated with Em were: age (ß 0.364; P=0.0001), GFR (beta 0.101; P=0.019), and the presence of diabetes (ß 0.166; P=0.002). Our study showed that in hypertensive patients with CKD the presence of diabetes is associated with increased LV-wall thicknesses and concentric geometry; further, diabetes together with renal function (GFR) is associated with worse diastolic function, independently of potential confounders, such as age, gender, body mass index and blood pressure.

Nitric oxide metabolites and oxidative stress in mild essential hypertension.

Many papers have showed non univocal data about oxidative stress status and nitric oxide metabolites in essential hypertension. Considering this preamble we examined the total antioxidant status (TAS), the lipid peroxidation (LP), expressed as thiobarbituric acid-reactive substances (TBARS), the stable end products of nitric oxide (NOx) and LP/NOx ratio in 25 subjects with untreated mild essential hypertension. The obtained data show a significant increase in TBARS (p < 0.001) and NOx (p < 0.001) in hypertensives and no variation in TAS and in TBARS/NOx ratio. None of these parameters was statistically related to the metabolic parameters or to the blood pressure values. The high level of lipid peroxidation observed in this group of hypertensives suggests the timely and specific employment of antihypertensive and antioxidant agents while the NOx increase seems to confirm the inflammatory status accompanying this clinical condition.