Reclamation and agricultural reuse of wastewater: the experience of the Cagliari sewage treatment plant (Sardinia, Italy).

In Sardinia, as in many other Mediterranean regions, recurrent droughts and climate change have dramatically reduced available water resources. As a result of this critical situation, in 1995 the Italian Government declared a state of emergency and drew up a program for financial support by the State and local authorities with the aim of reducing this serious deficit. One of the actions focused on reclaiming and reusing the effluent from the sewage treatment plant of Cagliari. This article reports on the multidisciplinary preliminary study performed by the Ente Acque della Sardegna (ENAS) to evaluate the suitability of reusing Is Arenas effluent for irrigation and on the operation of the tertiary treatment plant.

Stable transformation of erysiphe graminis an obligate biotrophic pathogen of barley.

Barley powdery mildew, Erysiphe graminis f.sp. hordei, is an obligate biotrophic pathogen and as such cannot complete its life cycle without a living host. The inability to transform this fungus and manipulate its genome has constrained research towards understanding its life cycle and pathogenicity. Here we describe an in planta transformation system based on delivery of DNA using a gold-particle gun and selection using benomyl or bialaphos. Using this method, we consistently obtained stable transformants with efficiencies comparable to other filamentous fungi. Stable expression of the beta-glucuronidase in E. graminis was demonstrated by co-transforming the uidA gene with the selectable markers.

The Apparent Turnover of 1-Aminocyclopropane-1-Carboxylate Synthase in Tomato Cells Is Regulated by Protein Phosphorylation and Dephosphorylation.

In suspension-cultured cells of tomato (Lycopersicon esculentum Mill.), the activity of 1-aminocyclopropane-1-carboxylate synthase (ACC-S) rapidly increases in response to fungal elicitors. The effect of inhibitors of protein kinases and protein phosphatases on the regulation of ACC-S was studied. K-252a, an inhibitor of protein kinases, prevented induction of the enzyme by elicitors and promoted its apparent turnover in elicitor-stimulated cells, causing a 50% loss of activity within 4 to 8 min in both the presence and absence of cycloheximide. Calyculin A, an inhibitor of protein phosphatases, caused a rapid increase of ACC-S in the absence of elicitors and an immediate acceleration of the rate of ACC-S increase in elicitor-stimulated cells. In the presence of cycloheximide there was no such increase, indicating that the effect depended on protein synthesis. Cordycepin, an inhibitor of mRNA synthesis, did not prevent the elicitor-induced increase in ACC-S activity but strongly reduced the K-252a-induced decay and the calyculin A-induced increase of its activity. In vitro, ACC-S activity was not affected by K-252a and calyculin A or by treatments with protein phosphatases. These results suggest that protein phosphorylation/dephosphorylation is involved in the regulation of ACC-S, not by regulating the catalytic activity itself but by controlling the rate of turnover of the enzyme.

Melatonin reduces the need for sedation in ICU patients: a randomized controlled trial.

BACKGROUND: Critically ill patients suffer from physiological sleep deprivation and have reduced blood melatonin levels. This study was designed to determine whether nocturnal melatonin supplementation would reduce the need for sedation in patients with critical illness. METHODS: A single-center, double-blind randomized placebo-controlled trial was carried out from July 2007 to December 2009, in a mixed medical-surgical Intensive Care Unit of a University hospital, without any form of external funding. Of 1158 patients admitted to ICU and treated with conscious enteral sedation, 82 critically-ill with mechanical ventilation >48 hours and Simplified Acute Physiology Score II>32 points were randomized 1:1 to receive, at eight p.m. and midnight, melatonin (3+3mg) or placebo, from the third ICU day until ICU discharge. Primary outcome was total amount of enteral hydroxyzine administered. RESULTS: Melatonin treated patients received lower amount of enteral hydroxyzine. Other neurological indicators (amount of some neuroactive drugs, pain, agitation, anxiety, sleep observed by nurses, need for restraints, need for extra sedation, nurse evaluation of sedation adequacy) seemed improved, with reduced cost for neuroactive drugs. Post-traumatic stress disorder prevalence did not differ between groups, nor did ICU or hospital mortality. Study limitations include the differences between groups before intervention, the small sample size, and the single-center observation. CONCLUSION: Long-term enteral melatonin supplementation may result in a decreased need for sedation, with improved neurological indicators and cost reduction. Further multicenter evaluations are required to confirm these results with different sedation protocols.

Use of monoclonal antibodies to determine biomass of Cladosporium fulvum in infected tomato leaves.

A monoclonal antibody, OX-CH1, was raised against surface washings of Cladosporium herbarum. This antibody recognizes an epitope that is found in various fungi belonging to the genus Cladosporium, including C. fulvum, the causal agent of tomato leaf mold. The epitope is present at comparable levels in two different races of C. fulvum and in transgenic isolates derived from them. The epitope is heat-and protease-resistant but sensitive to oxidation with periodate and it is constitutively expressed in C. fulvum grown in pure culture and on the plant. C. fulvum can be detected in infected tissues at levels starting from around 1 mg fresh weight of fungus per g fresh weight of leaf tissue. Noninfected tomato leaves do not cross-react with OX-CH1. We have developed an enzyme-linked immunosorbent assay (ELISA) for fungal biomass in tomato leaves and compared it with the assay based on measurements of beta-glucuronidase (GUS) activity in tissues infected with a transgenic isolate of C. fulvum race 4 carrying a uidA gene; the two assays give similar results.

Transcriptional regulation of components of the type III secretion system and effectors in Pseudomonas syringae pv. phaseolicola.

Quantitative real-time polymerase chain reaction was used with specific TaqMan probes to examine transcription of selected hrp and effector genes in Pseudomonas syringae pv. phaseolicola strains 1448A (race 6) and 1449B (race 7). Transcripts examined were from genes encoding the regulators hrpR and hrpL, core structural components of the type III secretion system (TTSS) hrcC, hrcJ, hrcN, hrcU, and hrpA; the first open-reading frame of each hrp operon, including hrpF, hrpJ, hrpP, and hrpY, and also secreted effectors hrpZ, avrPphE, avrPphF, and virPphA. All genes were induced by incubation in a minimal medium and showed patterns of expression indicating regulation by HrpRS and HrpL. Basal mRNA levels and the timing of accumulation of transcripts after induction differed significantly, suggesting the operation of additional regulatory elements. However, no clear transcriptional hierarchy emerged to explain the ordered construction of the TTSS. Quantitative analysis confirmed that the rates and levels of transcript accumulation within the first 2 h after inoculation were considerably higher in planta than in vitro, and indicated that plant cell wall contact may enhance transcription of TTSS and effector genes in P. syringae pv. phaseolicola. The low-abundance hrcU mRNA had a half-life of 16.5 min, whereas other transcripts had half-lives between 3 and 8 min.

HCF-1, a hydrophobin from the tomato pathogen Cladosporium fulvum.

A hydrophobin, named HCF-1, was isolated from the culture medium of Cladosporium fulvum, the causal agent of tomato leaf mould. The protein forms insoluble aggregates when the medium is vigorously aerated. These aggregates can be dissociated by trifluoroacetic acid into monomers which migrate as 10-kDa molecules on SDS-PAGE. HCf-1 is encoded by a single gene, HCf-1. The gene contains two small introns and is translated into a 105-amino acid protein which is then processed to give a mature 83-amino acid protein. The position of the eight cysteine residues and the predicted hydrophobicity profile are typical of fungal hydrophobins. HCf-1 RNA is expressed in growing mycelium and conidia but its quantity diminishes transiently after germination; its abundance does not change when the fungus is grown on nitrogen- or carbon-deficient medium. This is the first step in evaluating the role of hydrophobins in establishment of basic compatibility between C. fulvum and tomato.

HCf-6, a novel class II hydrophobin from Cladosporium fulvum.

C. fulvum, a fungal tomato pathogen, has previously been shown to express a complex family of hydrophobin genes including four class I hydrophobins and one class II hydrophobin. Here we describe a gene for HCf-6, a sixth member of the hydrophobin family and the second class II gene. The protein is predicted to consist of a signal sequence, an N-terminus rich in glycine and asparagine and a C-terminal hydrophobic domain which bears the hall-marks of hydrophobins. In contrast to the previously described class II hydrophobin HCf-5, HCf-6 is expressed in mycelium growing in pure culture and mRNA levels do not increase during sporulation. It is down-regulated by carbon starvation but not by depletion of nitrogen in the growth medium.

Different types of hysterectomy in the radio-surgical treatment of early cervical cancer (FIGO Ib-IIa).

PURPOSE OF INVESTIGATION: Surgery and radiotherapy achieved equivalent results for FIGO stages Ib-IIa invasive cervical carcinoma. The integration of radiotherapy and surgery provided the same results for a selected series of patients without increasing the rate of complications. The aim of the study was to verify if, applying a radio-surgical protocol, the reduction of the surgery extension on the parametrium in one of two consecutive series might achieve the same results in terms of survival and recurrence rates with fewer complications. MATERIAL AND METHODS: We analysed actuarial survival (with >10-year follow-up), local control rates and morbidity of 390 patients who had different the kinds of surgery applied in the radio-surgical treatment protocol: Protocol A: brachytherapy plus type III radical hysterectomy vs Protocol B: brachytherapy plus type II radical hysterectomy. Patients were all included in an analysis of complications according to the French-Italian glossary. RESULTS: Analyses showed no differences in terms of survival, patterns of recurrences and onset time within the two protocols. Urinary complications were more frequent and severe in protocol A vs protocol B (G2: 26.5% vs. 6.1%; G3: 5.3% vs. 3.6%). CONCLUSION: Our study pointed out that the reduction of the surgery extension allowed the same overall survival and relapses with fewer complications particularly in terms of grade of severity.

Aerophagia increases the risk of ventilator-associated pneumonia in critically-ill patients.

BACKGROUND: Gastric residual volume in ventilated critically ill may complicate gut function. Over the years studies suggested to tolerate progressively higher residuals. The relationship between such volumes and the development of ventilator-associated pneumonia (VAP) is still under debate. No reports deal with the relevant anecdotal finding of air in the stomach. Aim of the present study is to test the role of air in the development of VAPs. METHODS: Prospective observational trial in consecutive patients with a predicted length of ICU stay >3 days. The first 8 days of stay were studied. Sedation was targeted to have awake/cooperative patients. Early enteral nutrition was attempted. Gastric content was measured every 4 hours by 60 mL-syringe suction. Upper digestive intolerance (UDI) was defined as >2 consecutive findings of liquid >200 mL, aerophagia was defined as >2 consecutive findings of air >150 mL. RESULTS: Three hundred sixty-four patients enrolled, 43 developed VAP (11.8%). Patients were sedated with enteral (76% total time), intravenous (6%) or both (28%) drugs. Conscious sedation was achieved in 54% of the observations. 326 patients began enteral nutrition during the first 24 hours (1000 kcal median calorie intake). 10% developed UDI, 15% had aerophagia. No association was found between VAP and UDI (P=0.78), while significant association was found between VAP and aerophagia (OR=2.88, P<0.01). A sensitivity analysis, excluding patients admitted with respiratory infection, confirmed the results. CONCLUSION: High volumes of air in the stomach significantly increased the risk of developing VAP, while gastric residual volumes were not associated with the incidence of pneumonia.

Performance assessment of a glucose control protocol in septic patients with an automated intermittent plasma glucose monitoring device.

BACKGROUND & AIMS: The optimal level and modality of glucose control in critically ill patients is still debated. A protocolized approach and the use of nearly-continuous technologies are recommended to manage hyperglycemia, hypoglycemia and glycemic variability. We recently proposed a pato-physiology-based glucose control protocol which takes into account patient glucose/carbohydrate intake and insulin resistance. Aim of the present investigation was to assess the performance of our protocol with an automated intermittent plasma glucose monitoring device (OptiScanner™ 5000). METHODS: OptiScanner™ was used in 6 septic patients, providing glucose measurement every 15' from a side-port of an indwelling central venous catheter. Target level of glucose was 80-150 mg/dL. Insulin infusion and kcal with nutritional support were also recorded. RESULTS: 6 septic patients were studied for 319 h (1277 measurements); 58 [45-65] hours for each patient (measurements/patient: 231 [172-265]). Blood glucose was at target for 93 [90-98]% of study time. Mean plasma glucose was 126 ± 11 mg/dL. Only 3 hypoglycemic episodes (78, 78, 69 mg/dL) were recorded. Glucose variability was limited: plasma glucose coefficient of variation was 11.7 ± 4.0% and plasma glucose standard deviation was 14.3 ± 5.5 mg/dL. CONCLUSIONS: The local glucose control protocol achieved satisfactory glucose control in septic patients along with a high degree of safeness. Automated intermittent plasma glucose monitoring seemed useful to assess the performance of the protocol.

Intensive care of patients requiring open abdomen treatment: a case-series analysis.

BACKGROUND: This retrospective overview examines the management of patients with temporary open abdomen (OA). METHODS: The clinical characteristics and intensive care treatment of 34 consecutive patients with OA (1996-2012) were reviewed. RESULTS: Average age was 61 years, SAPS II score 43, SOFA 8. Two patients had non-contaminated abdomen; 12 had intact gut (only 8 later during stay); 7 repaired gut (only later 4); 13 cutaneous stoma (later 14), and 2 entero-atmospheric fistula (later 8+1 entero-enteral). The median ICU stay was 48 [36-94] days. One quarter of the 2376 ICU-days were classified as severe sepsis/septic shock (antibiotics were given for two thirds of the stay); three quarters were with ventilation; in 95% of days sedatives were given (mainly enterally). Continuous cavity lavage was done in three quarters of days; in 3% of days patients were fasted whereas >20 kcal/kg was given for 74% of days; we fed the gut in 95% of fed-days, in half of them combined with parenteral nutrition. Complications are discussed; mortality was 32.4%, limited to the ICU stay. CONCLUSION: The intensive care of patients with OA is challenging but can achieve better outcomes than expected. Continuous abdominal lavage improves the evacuation of contaminated fluid or debris and, coupled with antiseptics and low antibiotic pressure, reinforces the control of infection. The gut can be used for nutrition (even without gastrointestinal continuity), and long-term light sedation (mainly enteral) with minimal impact on perfusion, ventilation and gut motility.

Time course of endogenous nitric oxide inhibitors in severe sepsis in humans.

AIM: Asymmetric and symmetric dimethylarginines (ADMA and SDMA, respectively) are protein breakdown markers; both compete with arginine for cellular transport and both are excreted in urine. Moreover, ADMA is a non-selective inhibitor of nitric oxide (NO) synthase that is metabolized by a specific hydrolase in which the activity during stress remains controversial. While an increase in ADMA is known to be associated with adverse events, little is known about SDMA. We investigated plasma ADMA and SDMA levels during ICU stay to reveal the time course of endogenous NO inhibition in patients with sepsis. METHODS: A post hoc analysis from a prospective random controlled trial conducted in three ICUs was performed to study the pathophysiological pathways of sepsis. ADMA, SDMA, the ratio of ADMA/SDMA (a marker of ADMA catabolism), arginine, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and C reactive protein (CRP) were measured on days 1, 3, 6, 9, 12 and at discharge in 72 consecutive severely septic patients. RESULTS: Fasting basal glycemia, creatinine, IL-6, TNF-alpha, CRP, ADMA, and SDMA were higher than normal. The ADMA/SDMA ratio was decreased by 50%, and arginine levels were low. ADMA levels were related to the total Sequential Organ Failure Assessment (SOFA) scores and arginine levels, and inversely related to IL-6 and CRP levels. SDMA levels were related to Simplified Acute Physiologic Scores II (SAPS II), SOFA scores, blood urea, creatinine, and arginine levels. The ADMA/SDMA ratio was inversely related to IL-6 levels. In 58 ICU survivors, creatinine, IL-6, and CRP levels decreased over time; ADMA levels increased, SDMA levels remained stable, and the ADMA/SDMA ratio increased. In 14 non-survivors, creatinine, IL-6, TNF-alpha, CRP, and ADMA levels were stable, whereas the SDMA levels increased and the ADMA/SDMA ratio remained low. In both ICU survivors and non-survivors, the levels on the last ICU day confirmed the data trends. SDMA, but not ADMA, was associated with ICU mortality. CONCLUSION: ADMA catabolism appears to be activated by inflammation; its increase during the advanced septic phase in surviving patients may suggest an endogenous inhibition of NO synthesis during the full-blown septic phase. In severe sepsis, SDMA, but not ADMA, appears to be a marker of alterations in vital functions and mortality.

Effects of recombinant human activated protein C on the fibrinolytic system of patients undergoing conventional or tight glycemic control.

AIM: Recombinant human activated protein C (rh-APC) and tight glycemic control (TGC) have been shown to reduce mortality in septic patients. Both interventions can reduce the plasma concentration and/or activity of the most powerful suppressor of fibrinolysis, plasminogen activator inhibitor-1 (PAI-1). Our aim was to evaluate the effects on the fibrinolytic system after the administration of rh-APC in septic patients undergoing conventional or TGC. METHODS: Posthoc analysis of data was collected from 90 patients with severe sepsis/septic shock, randomized to either conventional or TGC groups. Independent of these treatments, patients with at least two organ dysfunctions simultaneously received rh-APC. Plasma levels of multiple biochemical markers for fibrinolysis, coagulation, and inflammation were determined every day for the 1st week and then on study days 9, 11, 13, 18, 23, and 28. Clinical data and sepsis-related organ failure assessment (SOFA) scores were also recorded. RESULTS: Patients who had received rh-APC exhibited significantly more impairments in fibrinolysis at baseline (PAI-1 activity 49.76 [24.61-71.82] vs 21.92 [6.47-55-83] IU/mL, P=0.03). The reductions in plasma PAI-1 activity over time associated with rh-APC treatment were different according to whether the treatment was administered to patients undergoing conventional or TGC (P=0.01). However, the most prominent reductions were in patients undergoing conventional glycemic control. Significant interactions between the two study interventions were also found for PAI-1 concentration (P<0.001), C-reactive protein (P=0.02), and interleukin-6 levels (P<0.001). CONCLUSIONS: Both rh-APC and TGC appear to improve fibrinolysis in septic patients. The reduction in the impairment of fibrinolysis associated with rh-APC treatment seems greater in patients undergoing conventional glycemic control than in those undergoing TGC.

Metabolic treatment of critically ill patients: energy balance and substrate disposal.

Oxidation of substrates is the main biochemical process used by the human body to produce energy. Different substrates (carbohydrates, lipids, and proteins) have different effects on oxygen consumption and carbon dioxide production: during the critical phase of pathologies it could be relevant pay attention to the use of various nutrients, that have some altered effect respect to the normal subjects metabolism, and during the length of metabolic treatment, too. Generally, nutrition lead to replenish body stores, while endogenous substrates are used to be oxidized. Critically ill patients show a preference for prompt energy availability (i.e. glucose) to avoid endogenous protein catabolism; lipids are shown to have a more pronounced storage effect. Adequate amount of energy intake in carbohydrates determine an increase of RQ, that means a shift from a more lipid-based to a more glucose-based oxidation. Composition of dietary intake can be usefully different for each pathology, and also for different periods of the same pathology, because critically ill patients have a variety of metabolic needs during their stay in ICU.

Analysis and cloning of the ethylene-forming enzyme from tomato by functional expression of its mRNA in Xenopus laevis oocytes.

The last step in biosynthesis of the plant hormone ethylene, oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC), is catalysed by the elusive ethylene-forming enzyme (EFE). EFE is induced by fungal elicitors in suspension-cultured tomato cells. We demonstrate that Xenopus laevis oocytes injected with RNA from elicitor-treated tomato cells gain the ability to convert ACC to ethylene. The enzyme expressed in the oocytes under the direction of plant RNA is indistinguishable from genuine plant EFE with regard to its saturation kinetics, its iron dependency and its stereospecificity to the diastereomeric ethyl derivatives of ACC, allocoronamic acid and coronamic acid. In tomato cells stimulated for different times with elicitor, the level of EFE correlates with the level of RNA directing EFE expression in oocytes. Hybridization and co-injection experiments demonstrate that the tomato RNA species directing EFE expression in oocytes are homologous to clone pTOM13 which has been shown to inhibit ethylene production in plants when expressed in antisense. Using a cDNA library from elicitor-stimulated tomato cells, we have isolated several homologues of pTOM13 and identified one of them, pHTOM5, as a clone of EFE on the basis of its functional expression in the Xenopus oocytes.

Co-suppression of the hydrophobin gene HCf-1 is correlated with antisense RNA biosynthesis in Cladosporium fulvum.

Transformation of Cladosporium fulvum with DNA containing a truncated copy of the hydrophobin gene HCf-1 causes co-suppression of hydrophobin synthesis in 30% of the transformants. The co-suppressed isolates have a hydrophilic phenotype, lower levels of HCf-1 mRNA than wild type and contain multiple copies of the plasmid integrated as tandem repeats at ectopic sites in the genome. Gene silencing is not associated with DNA cytosine methylation. Nuclear run-off experiments reveal that transcription rate of HCf-1 in the co-suppressed isolates is higher than in the untransformed strains, suggesting that silencing acts at the post-transcriptional level. We show, for the first time in fungi, that co-suppression is correlated with the presence of antisense RNA, and that this is synthesised on a DNA template. Derivatives showing reversion to the wild-type phenotype and restoration of HCf-1 gene expression were also observed. Reversion is associated with loss of some copies of the transgene. We propose that co-suppression is due to ectopic integration of the transgene next to promoters which initiate transcription to form antisense RNA and that this in turn determines down-regulation of HCf-1.

Inactivation of stress induced 1-aminocyclopropane carboxylate synthase in vivo differs from substrate-dependent inactivation in vitro.

The activity of 1-aminocyclopropane carboxylate (ACC) synthase increased rapidly in tomato (Lycopersicon esculentum Mill.) leaf discs after vacuum infiltration, reached a maximum after about 30 minutes, and subsequently decayed with an apparent half-life of about 20 minutes. Aminoethoxyvinylglycine, a known inhibitor of ACC synthase, did not alter the apparent turnover of ACC synthase in vivo although it efficiently blocked inactivation of the enzyme by its substrate S-adenosylmethionine in vitro. Similar results were obtained, using a novel assay with permeabilized cells, for ACC synthase in tomato cell cultures treated with a fungal elicitor. The results indicate that inactivation of ACC synthase in vivo differs from substrate-dependent inactivation in vitro.