Industrial scale bio-detoxification of raw olive mill wastewaters by the use of selected microbial yeast and bacterial strains to obtain a new source for fertigation.

The Olive Mill Wastewaters (OMWs) are one of the most important agro-industrial wastes of the Mediterranean Countries and the disposal by draining them onto land has been proved to be damaging for soils, plants and groundwater due to their polluting power. The present report describes a new method for bio-detoxification of undiluted fresh OMW based on the driven selection of aerobic yeasts and bacteria. The identified yeast Candida boidinii A5y and the bacterium Paenibacillus albidus R32b strains allowed the treatment of freshly produced raw OMW characterized by very high COD value and phenolic content, when applied as sequential inoculum. The treated OMW showed the absence of antimicrobial effects and a strongly reduction of phytotoxic activity on the germination of several plant seeds. The process was successfully validated on an industrial scale without any pre-treatment, dilution and/or supplementation of the raw waste. Bio-detoxified OMW produced by this sustainable and low-cost process would be suitable for new non-chemical fertigation or soilless applications. The described procedure represents a virtuous example of circular economy efficaciously applied for a depleting agri-food resource.

Sleep-disordered breathing and epicardial adipose tissue in patients with heart failure.

BACKGROUND AND AIMS: Sleep-disordered breathing (SDB) is common in patients with heart failure (HF), contributes to the progression of cardiac disease, and is associated with adverse prognosis. Previous evidence indicates that epicardial adipose tissue (EAT) is independently associated with sleep apnea in obese individuals. We explored the relationship between SDB and EAT in HF patients. METHODS AND RESULTS: EAT thickness was assessed by echocardiography in 66 patients with systolic HF undergoing nocturnal cardiorespiratory monitoring. A significantly higher EAT thickness was found in patients with SDB than in those without SDB (10.7 ± 2.8 mm vs. 8.3 ± 1.8 mm; p = 0.001). Among SDB patients, higher EAT thickness was found in both those with prevalent obstructive sleep apnea (OSA) and those with prevalent central sleep apnea (CSA). Of interest, EAT thickness was significantly higher in CSA than in OSA patients (11.9 ± 2.9 vs. 10.1 ± 2.5 p = 0.022). Circulating plasma norepinephrine levels were higher in CSA than in OSA patients (2.19 ± 1.25 vs. 1.22 ± 0.92 ng/ml, p = 0.019). According to the apnea-hypopnea index (AHI), patients were then stratified in three groups of SDB severity: Group 1, mild SDB; Group 2, moderate SDB; Group 3, severe SDB. EAT thickness progressively and significantly increased from Group 1 to Group 3 (ANOVA p < 0.001). At univariate analysis, only left ventricular ejection fraction and AHI significantly correlated with EAT (p = 0.019 and p < 0.0001, respectively). At multivariate analysis, AHI was the only independent predictor of EAT (ß = 0.552, p < 0.001). CONCLUSIONS: Our results suggest an association between the presence and severity of sleep apneas and cardiac visceral adiposity in HF patients.

A local glucagon-like peptide 1 (GLP-1) system in human pancreatic islets.

AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) is a major incretin, mainly produced by the intestinal L cells, with beneficial actions on pancreatic beta cells. However, while in vivo only very small amounts of GLP-1 reach the pancreas in bioactive form, some observations indicate that GLP-1 may also be produced in the islets. We performed comprehensive morphological, functional and molecular studies to evaluate the presence and various features of a local GLP-1 system in human pancreatic islet cells, including those from type 2 diabetic patients. METHODS: The presence of insulin, glucagon, GLP-1, proconvertase (PC) 1/3 and PC2 was determined in human pancreas by immunohistochemistry with confocal microscopy. Islets were isolated from non-diabetic and type 2 diabetic donors. GLP-1 protein abundance was evaluated by immunoblotting and matrix-assisted laser desorption-ionisation-time of flight (MALDI-TOF) mass spectrometry. Single alpha and beta cell suspensions were obtained by enzymatic dissociation and FACS sorting. Glucagon and GLP-1 release were measured in response to nutrients. RESULTS: Confocal microscopy showed the presence of GLP-1-like and PC1/3 immunoreactivity in subsets of alpha cells, whereas GLP-1 was not observed in beta cells. The presence of GLP-1 in isolated islets was confirmed by immunoblotting, followed by mass spectrometry. Isolated islets and alpha (but not beta) cell fractions released GLP-1, which was regulated by glucose and arginine. PC1/3 (also known as PCSK1) gene expression was shown in alpha cells. GLP-1 release was significantly higher from type 2 diabetic than from non-diabetic isolated islets. CONCLUSIONS/INTERPRETATION: We have shown the presence of a functionally competent GLP-1 system in human pancreatic islets, which resides in alpha cells and might be modulated by type 2 diabetes.

Immunology in the clinic review series; focus on type 1 diabetes and viruses: how viral infections modulate beta cell function.

Type 1 diabetes mellitus (T1DM) is a multi-factorial immune-mediated disease characterized by the autoimmune destruction of insulin-producing pancreatic islet beta cells in genetically susceptible individuals. Epidemiological evidence has also documented the constant rise in the incidence of T1DM worldwide, with viral infections representing one of the candidate environmental risk factors identified by several independent studies. In fact, epidemiological data showed that T1DM incidence increases after epidemics due to enteroviruses and that enteroviral RNA can be detected in the blood of >50% of T1DM patients at the time of disease onset. Furthermore, both in-vitro and ex-vivo studies have shown that viruses can infect pancreatic beta cells with consequent effects ranging from functional damage to cell death.

Delta-cell-specific expression of hedgehog pathway Ptch1 receptor in murine and human endocrine pancreas.

BACKGROUND: Hedgehog pathway plays an important role during pancreas development, when its inactivation is crucial to assure expression of pancreatic marker genes involved in the organ formation and to assure an appropriate organogenesis. Patched1 (Ptch1) is a transmembrane receptor of hedgehog pathway which has a key role in this process. In fact, heterozygous Ptch1 mutant (ptc+/-) mice are affected by an impaired glucose tolerance accompanied by reduced islet function. In the light that the cell distribution of Ptch1 receptor within the endocrine pancreas has not yet been established, we aimed at identifying the pancreatic endocrine cell subset(s) expressing such molecule. METHODS: Double immunostaining for Ptch1 and pancreatic hormones insulin, glucagon and somatostatin on pancreatic paraffin sections of C57BL/6J mice and human non-diabetic multiorgan donors was performed and analysed using confocal microscopy. In addition, diabetes was experimentally induced in mice by intraperitoneal injection of streptozotocin. Quantitative real-time polymerase chain reaction after laser-capture microdissection of different islets from frozen pancreatic murine tissue sections was also performed. RESULTS: Ptch1 receptor was detected only in somatostatin-positive delta cells both in mice and in human pancreas; in mice its expression was not affected by streptozotocin treatment. A significant increase of Ptch1 mRNA expression levels in the islet periphery versus the islet core was observed by quantitative real-time polymerase chain reaction, in accord with immunohistochemical observations. CONCLUSION: Our data show a delta-cell-specific expression of Ptch1 receptor in murine and human pancreas.

Expression of Lactobacillus brevis IOEB 9809 tyrosine decarboxylase and agmatine deiminase genes in wine correlates with substrate availability.

AIMS: Lactobacillus brevis IOEB 9809 is able to produce both tyramine and putrescine via tyrosine decarboxylase and agmatine deiminase enzymes, respectively, when cultured on synthetic media. The aims of this study were to assess the expression of L. brevis IOEB 9809 tdc and aguA1 genes, during wine fermentation and to evaluate the effect of substrate availability and pH on tdc and aguA1 expression, as well as on biogenic amine production and L. brevis viability. METHODS AND RESULTS: The relative expression of L. brevis IOEB 9809 tdc and aguA1 genes was analysed in wine by quantitative real-time RT-PCR (qRT-PCR) during a period of incubation of 30 days. Cell viability, pH values, putrescine and tyramine concentration were monitored throughout the experiments. CONCLUSIONS: The wine trials indicated that L. brevis IOEB 9809 is able to produce both tyramine and putrescine during wine fermentation. Increased cell viability was also observed in wine supplemented with tyrosine or agmatine. qRT-PCR analysis suggests a strong influence of substrate availability on the expression of genes coding for tyrosine decarboxylase and agmatine deiminase in L. brevis IOEB 9809. Less evident is the relationship between putrescine and tyramine production and tolerance to wine pH. SIGNIFICANCE AND IMPACT OF STUDY: To our knowledge, this study represents the first assessment of relative expression of L. brevis IOEB 9809 genes involved in biogenic amine production in wine. Furthermore, an effect of biogenic amine production on viability of L. brevis during wine fermentation was established.

Palmitate induces a pro-inflammatory response in human pancreatic islets that mimics CCL2 expression by beta cells in type 2 diabetes.

AIMS/HYPOTHESIS: Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate. METHODS: Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes. RESULTS: Microarray analysis of laser-capture microdissected beta cells showed increased chemokine and cytokine expression in type 2 diabetes compared with non-diabetic controls. The inflammatory response in type 2 diabetes was mimicked by exposure of non-diabetic human islets to palmitate, but not to oleate or high glucose, leading to the induction of IL-1beta, TNF-alpha, IL-6, IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2). Interference with IL-1beta signalling abolished palmitate-induced cytokine and chemokine expression but failed to prevent lipotoxic human islet cell death. Palmitate activated nuclear factor kappaB (NF-kappaB) in human pancreatic beta and non-beta cells, and chemically induced endoplasmic reticulum stress caused cytokine expression and NF-kappaB activation similar to that occurring with palmitate. CONCLUSIONS/INTERPRETATION: Saturated-fatty-acid-induced NF-kappaB activation and endoplasmic reticulum stress may contribute to IL-1beta production and mild islet inflammation in type 2 diabetes. This inflammatory process does not contribute to lipotoxicity ex vivo, but may lead to local chemokine release.

Technological properties of Oenococcus oeni strains isolated from typical southern Italian wines.

AIMS: To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach. METHODS AND RESULTS: Oenococcus oeni strains were isolated from Nero di Troia wines undergoing spontaneous MLF. Samples were taken at the end of alcoholic fermentation and during MLF. Wine samples were diluted in a sterile physiological solution and plated on MRS and on modified FT80. Identification of O. oeni strains was performed by a polymerase chain reaction (PCR) experiment using strain-specific primers. Strains were further grouped using a multiplex RAPD-PCR analysis. Then, six strains were inoculated in two winelike media with two different ethanol concentrations (11 and 13% vol / vol) with a view to evaluate their capacity to grow and to perform MLF. In addition, a quantitative PCR (qRT-PCR) approach was adapted to monitor the physiological state of the strains selected. CONCLUSION: A positive correlation between the malolactic activity performance and the ability to develop and tolerate stress conditions was observed for two selected O. oeni strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The results reported are useful for the selection of indigenous MLF starter cultures with desired oenological traits from typical regional wines. It should be the base for the improvement in organoleptic quality of typical red wine.

Genotyping by Amplified Fragment Length Polymorphism and malate metabolism performances of indigenous Oenococcus oeni strains isolated from Primitivo wine.

The Amplified Fragment Length Polymorphism (AFLP) technique was applied for the first time to investigate the genotyping of Oenococcus oeni, the most important species involved in malolactic fermentation (MLF) in wine. A total of 87 out of 220 lactic acid bacteria, isolates from "Primitivo" wine (Apulia, Italy) undergoing MLF, identified as O. oeni by species-specific PCR and 16S rRNA sequence analysis, were studied by AFLP analysis. Four main clusters were distinguished and three of them showed intraspecific homology higher than 60%. A total of 28 strains, representative of AFLP clusters, were tested for malate metabolism in order to gain information on their malolactic performances. Significant differences were observed among strains for malic acid consumed, biomass produced and specific malic acid consumption rate. These findings indicated that AFLP technique is reliable for typing O. oeni strains and that, together with metabolism studies it may be used to individuate possible candidates as industrial malolactic starters.

Differential expression of neurogenins and NeuroD1 in human pituitary tumours.

Basic helix-loop-helix (bHLH) transcription factors are involved in neuroendocrine cell growth and differentiation. Though NeuroD1 is viewed as corticotroph specific, its overexpression in non-corticotroph pituitary adenomas (PAs) may reflect the activation of molecular pathways involving other bHLH factors, like neurogenins. To search for neurogenin-NeuroD1 molecular pathways in the human normal and tumoural pituitary. Fifty-one PAs--22 clinically non-secreting (CNS) and 29 secreting respectively--and normal human pituitaries (NP) were studied for NeuroD1 and neurogenins (Ngn1, Ngn2 and Ngn3) gene expression by RT-PCR and quantitative real-time RT-PCR (qRT-PCR). Immunohistochemistry for Ngn2/3 was performed in some cases. NeuroD1, Ngn2, Ngn3 and Ngn1 were observed in up to 84.3, 76.5, 30.4 and 9.1% of PA respectively, only NeuroD1 and Ngn2 being frequently overexpressed when compared with NP. Whereas NeuroD1 expression was higher in corticotroph and CNS adenomas (P=0.0001 versus Pit-1-dependent PA), Ngn2 expression was higher in secreting PA, especially in Pit-1-dependent PA (P=0.007 and P=0.0006 versus CNS respectively). Pit-1-dependent PA which received pre-operative pharmacological treatment expressed higher Ngn2 levels than untreated cases (P=0.025). Nuclear Ngn2 was observed in NP and in most PA, especially ACTH- and GH-secreting adenomas. Nuclear Ngn3 was observed in a minority of secreting PA. Ngn2 is normally expressed in the anterior pituitary and frequently expressed in PA, but does not account for NeuroD1 overexpression where present. Owing to their low and inconstant expression, the biological significance of Ngn1/3 in the adult pituitary is uncertain.

Histamine: a putative afferent neurotransmitter in Limulus eyes.

Histamine has been proposed as a photoreceptor neurotransmitter in two major groups of arthropods, the insects and the crustacea. In this study biochemical and immunocytochemical approaches were used to examine the synthesis, endogenous content, and cellular distribution of histamine in the visual system of the horseshoe crab Limulus polyphemus, an ancient chelicerate arthropod. Studies with this animal have been critical to our understanding of the basic processes of vision. High-voltage paper electrophoresis was used to assay for histamine synthesis in Limulus tissues incubated with radiolabeled histidine; histamine synthesis was detected in the lateral, median, and ventral eyes and optic nerves and in the visual centers in the brain. Endogenous histamine, assayed as its orthophthalaldehyde derivative by high-performance liquid chromatography and electrochemical detection, was also detected in these tissues. Immunocytochemical analyses, with an antiserum directed against a protein conjugate of histamine, revealed histamine-like immunoreactivity in the somata of photoreceptors in each of the eyes and in the regions of the brain where the photoreceptors terminate. Histamine-like immunoreactivity was also intense in the cell bodies and axon collaterals of eccentric cells in the lateral eye and in eccentric cell projections in the brain. These results show that histamine is a major biogenic amine in the Limulus visual system, and they suggest that this amine is involved in transmitting visual information from the eyes to the brain and in lateral inhibition, a fundamental mechanism for processing visual information in the lateral eye.

Osteonecrosis of the femoral head.

New cases of osteonecrosis of the femoral head in the United States number between 10,000 and 20,000 per year. This disease usually affects patients in their late 30s and early 40s. Although a number of authors have related specific risk factors to this disease, its etiology, pathogenesis, and treatment remain a source of considerable controversy. This disorder has been associated with corticosteroid use, substance abuse, and various systemic medical conditions. Either direct damage to osteocytes (e.g., by toxin production) or indirect damage (e.g., due to disorders in fat metabolism or hypoxia) may lead to osteonecrosis. Patients at increased risk for osteonecrosis should be monitored closely. Unfortunately, most cases are diagnosed in an advanced stage of disease, when minimally invasive surgical procedures are no longer helpful. Furthermore, patients in the advanced stage of the disease must undergo total hip replacement at a young age, which carries a poor long-term prognosis.

USP18 is a key regulator of the interferon-driven gene network modulating pancreatic beta cell inflammation and apoptosis.

Type 1 diabetes (T1D) is an autoimmune disease targeting pancreatic beta cells. Genome-wide association studies and gene expression analysis identified interferon (IFN)-driven gene networks as crucial pathways in the pathogenesis of T1D. IFNs are linked to the response to viral infections and might contribute to the initiation of the autoimmune process in T1D. We presently analyzed the role of ubiquitin-specific peptidase 18 (USP18), an interferon-stimulated gene 15-specific protease, on IFN-induced pancreatic beta cell inflammation and apoptosis. Our findings indicate that USP18 inhibition induces inflammation by increasing the STAT signaling and exacerbates IFN-induced beta cell apoptosis by the mitochondrial pathway of cell death. USP18 regulates activation of three BH3-only proteins, namely, DP5, Bim and PUMA in pancreatic beta cells, suggesting a direct link between regulators of the type I IFN signaling pathway and members of the BCL-2 family. USP18 depletion increases the expression of the T1D candidate gene MDA5, leading to an upregulation of double-stranded RNA-induced chemokine production. These data suggest a cross talk between the type I IFN signaling pathway and a candidate gene for T1D to increase pro-inflammatory responses in beta cells. The present study shows that USP18 is a key regulator of IFN signaling in beta cells and underlines the importance of this pathway in beta cell inflammation and death.

Selection of non-conventional yeasts and their use in immobilized form for the bioremediation of olive oil mill wastewaters.

The yeast population dynamics in olive wastewaters (OMW), sampled in five mills from Salento (Apulia, Southern Italy), were investigated. Three hundred yeasts were isolated in five industrial mills and identified by molecular analysis. Strains belonging to Geotrichum, Saccharomyces, Pichia, Rhodotorula and Candida were detected. Five G. candidum strains were able to grow in OMW as the sole carbon source and to reduce phenolics, chemical oxygen demand (COD) and antimicrobial compounds. One G. candidum isolate was selected for whole-cell immobilization in calcium alginate gel. The COD and phenolic reduction obtained with immobilized cells showed a 2.2- and 2-fold increase compared to the removal obtained with free cells, respectively. The immobilization system enhanced yeast oxidative activity by avoiding the presence of microbial protease in treated OMW. To our knowledge, this is the first report on G. candidum whole-cell immobilization for OMW bioremediation.

The role of the C-terminal region of olive latent virus 1 coat protein in host systemic infection.

A full-length cDNA clone of olive latent virus 1 (OLV-1), a member of the genus Necrovirus, family Tombusviridae, was subjected to site-directed mutagenesis, and coat protein gene mutants were constructed. A mutant clone, denoted Delta3297, was obtained by deleting the nucleotide in position 3297, thus inducing a frameshift and replacing the last 49 amino acids of the viral coat protein (CP) by a shorter sequence of 39 amino acids. This mutant was viable, stable, able to synthesize a smaller CP, and able to give rise to the formation of apparently intact virus particles. Cell-to-cell movement of Delta3297 in Nicotiana benthamiana leaves was not affected, but, contrary to wild type OLV-1, it failed to spread systemically. These results indicate that virion formation is necessary but not sufficient for long-distance movement for OLV-1 and highlights the role of the CP carboxy-terminal domain in systemic infection.

Persistence of fenitrothion in oranges and clementines after treatment with emulsifiable concentrate and microencapsulate formulations.

The rate of decline of fenitrothion residues was investigated in oranges and clementines after treatment with two different kinds of commercial formulations: emulsifiable concentrate (Afidina M) and microencapsulate (Fenitrocap and IPM 400). The study was performed on the fruit and leaves over 131 and 161 days for oranges, and over 78 and 86 days for clementines, respectively. In fruit, the experimental data showed a similar behaviour of the active ingredient for both kinds of commercial formulations. High mean levels of fenitrothion (between about 0.4 and 0.8 mg kg(-1)) were persistent for at least 75 days after treatment in oranges and 50 days in clementines, with statistically significant declines observed only at days 110 and 78, respectively. A rapid decline of fenitrothion levels was observed in orange and clementine leaves during the starting phase followed by a slower decrease during the later stage; the decline was more pronounced with the treatment of emulsifiable concentrates. These findings are indicative of a poor degradability of fenitrothion in citrus fruits, and suggest that repeated or uneven applications of the pesticide should be avoided in order to exclude the risk of exceeding the maximum residue level permitted by the current regulations.

Subcellular localization and immunodetection of movement proteins of olive latent virus 1.

Polyclonal sera raised to Escherichia coli-expressed movement proteins encoded by ORF 3 (p8K) and ORF 4 (p6K) of olive latent virus 1, were used for their immunodetection in infected Nicotiana benthamiana plants. In subfractionated locally infected tissues 4 days post inoculation (d.p.i.) that were analysed by Western blot, p8K was found in the fast-sedimenting fractions P1 and P30 containing membranous material and/or cell organelles and, likely, the fibrous structures mentioned below, but not in the soluble protein-containing supernatant. No p6K could be detected in these extracts. In locally inoculated leaves p8K began to accumulate from 2 d.p.i onwards reaching its peak at 4 d.p.i. Intracellular immunogold labelling of cells from locally and systemically infected tissues localized p8K primarily in fibrous inclusions made up of thin filaments with a helical structure present in the cytoplasm of locally and systemically infected cells. In systemic infections a light and scattered labelling was observed in the cytoplasm and near the cell wall. The specific serum to p6K did not label the fibrous structures and failed to recognize its antigen in systemically and locally infected tissues except at 4 d.p.i., when scattered labelling was observed in the cytoplasm and near plasmodesmata.

Evolutionary dynamics of cucumber mosaic virus satellite RNA during natural epidemics in Italy.

The evolutionary dynamics of 22 variants of cucumber mosaic virus satellite RNA (CMV satRNA) isolated in Italy during virus epidemics from 1988 to 1993 were investigated on the basis of their primary structure and biological properties. Most of the variants were amplified from total nucleic acid preparations extracted from field-infected plants, thus representing wild isolates of CMV satRNA. Eleven variants were associated with subgroup II CMV strains, 10 with subgroup I and 1 with a mixed infection by both strains. When inoculated onto tomato seedlings, the variants induced the phenotype (necrogenic or ameliorative) predicted by their nucleotide sequence. Phylogenetic relationships between the satRNA variants were determined using the stationary Markov model, a stochastic model for evolution. For each satRNA, the Markov analysis gave a good correlation between position in the phylogenetic tree and biological properties. The variants with ameliorative and necrogenic phenotypes in tomato followed two different evolutionary dynamics in nature. Tfn-satRNA, a 390-nt-long molecule, followed a third type of evolutionary dynamic far apart from that of the shorter satRNA molecules (i.e., those in the 334- to 340-nt-length class). Average values of the mean constant rate of nucleotide substitutions/site (Ksubs/site) indicated that in nature the variants tend to keep their heterogeneity unchanged from one epidemic episode to the other, even if the outbreaks occur in places very far from each other. This seems to be in agreement with the proposed maintenance of a functional molecular structure as a constraint to CMV satRNA evolution.

Nucleotide sequence of the 3'-terminal region of cymbidium ringspot virus RNA.

The nucleotide sequences of cDNA clones corresponding to 2569 nucleotides from the 3' end of cymbidium ringspot tombusvirus (CyRSV) RNA were determined. This region contains three open reading frames giving rise to three predicted protein products, two of which had been identified in previous studies. The 3' non-coding region is 351 nucleotides long. The amino acid sequence of CyRSV coat protein has striking similarities with that of tomato bushy stunt tombusvirus, particularly in the S domain. No homology was found between the protein encoded by the second largest open reading frame and the corresponding product of other plant viruses.