Life Cycle Assessment of a system for the extraction and transformation of Waste Water Treatment Sludge (WWTS)-derived lipids into biodiesel.

In recent years, the demand for biofuels has been growing exponentially, as has the interest in biodiesel produced from organic matrices. Particularly interesting, due to its economic and environmental advantages, is the use of the lipids present in sewage sludge as a raw material for the synthesis of biodiesel. The possible processes of this biodiesel synthesis, starting from lipid matter, are represented by the conventional process with sulfuric acid, by the process with aluminium chloride hexahydrate and by processes that use solid catalysts such as those consisting of mixed metal oxides, functionalized halloysites, mesoporous perovskite and functionalized silicas. In literature there are numerous Life Cycle Assessment (LCA) studies concerning biodiesel production systems, but not many studies consider processes that start from sewage sludge and that use solid catalysts. In addition, no LCA studies were reported on solid acid catalysts nor on those based on mixed metal oxides which present some precious advantages, over the homogeneous analogous ones, such as higher recyclability, prevention of foams and corrosion phenomena, and an easier separation and purification of biodiesel product. This research work reports the results of a comparative LCA study applied to a system that uses a solvent free pilot plant for the extraction and transformation of lipids from sewage sludge via seven different scenarios that differ in the type of catalyst used. The biodiesel synthesis scenario using aluminium chloride hexahydrate as catalyst has the best environmental profile. Biodiesel synthesis scenarios using solid catalysts are worse due to higher methanol consumption which requires higher electricity consumption. The worst scenario is the one using functionalized halloysites. Further future developments of the research require the passage from the pilot scale to the industrial scale in order to obtain environmental results to be used for a more reliable comparison with the literature data.

Investigating the population structure and genetic differentiation of livestock guard dog breeds.

Livestock guarding dogs are a valuable adjunct to the pastoral community. Having been traditionally selected for their working ability, they fulfil their function with minimal interaction or command from their human owners. In this study, the population structure and the genetic differentiation of three Italian livestock guardian breeds (Sila's Dog, Maremma and Abruzzese Sheepdog and Mannara's Dog) and three functionally and physically similar breeds (Cane Corso, Central Asian Shepherd Dog and Caucasian Shepherd Dog), totalling 179 dogs unrelated at the second generation, were investigated with 18 autosomal microsatellite markers. Values for the number of alleles per locus, observed and expected heterozygosity, Hardy-Weinberg Equilibrium, F stats, Nei's and Reynold's genetic distances, clustering and sub-population formation abilities and individual genetic structures were calculated. Our results show clear breed differentiation, whereby all the considered breeds show reasonable genetic variability despite small population sizes and variable selection schemes. These results provide meaningful data to stakeholders in specific breed and environmental conservation programmes.

Supplementation of Rosemary extract in the diet of Nero Siciliano pigs: evaluation of the antioxidant properties on meat quality.

In the present study, the effect of Rosmarinus officinalis L. dietary supplementation on meat quality and oxidative stability of Nero Siciliano pigs was examined. During the growing-fattening period, 32 Nero Siciliano pigs were allotted into two treatment groups consisting of 8 replicates with 2 pigs per pen. For 90 days, the animals received a basal diet: one group (CTR) was not dietary supplemented, whereas the other group received (1 g/kg) rosemary extract (ROX). Supplementation with rosemary extract significantly improved the polyunsaturated fatty acid content of the meat, which showed higher values in the meat of the ROX group compared with the CTR group (P0.05). Color measurement performed in the present study on meat samples from the two dietary treatments showed that redness decreased (P=0.046) and hue values increased (P=0.036), indicating that a deterioration of the initial color occurred and that the rosemary extract was ineffective in preventing color deterioration. Nevertheless, the lightness, yellowness and chroma color descriptors showed similar values in relation to dietary treatment (P>0.05). Considering the nutritional value of meat as an important contributor to the overall quality, the results obtained in this study support the possibility of the dietary supplementation with R. officinalis L. extract in pigs as a functional additive in livestock feeding.

A cross-sectional nationwide survey on esophageal atresia and tracheoesophageal fistula.

BACKGROUND: Our study aims at disclosing epidemiology and most relevant clinical features of esophageal atresia (EA) pointing to a model of multicentre collaboration. METHODS: A detailed questionnaire was sent to all Italian Units of pediatric surgery in order to collect data of patients born with EA between January and December 2012. The results were crosschecked by matching date and place of birth of the patients with those of diagnosis-related group provided by the Italian Ministry of Health (MOH). RESULTS: A total of 146 questionnaires were returned plus a further 32 patients reported in the MOH database. Basing on a total of 178 patients with EA born in Italy in 2012, the incidence of EA was calculated in 3.33 per 10,000 live births. Antenatal diagnosis was suspected in 29.5% patients. 55.5% showed associated anomalies. The most common type of EA was Gross type C (89%). Postoperative complications occurred in 37% of type C EA and 100% of type A EA. A 9.5% mortality rate was reported. CONCLUSIONS: This is the first Italian cross-sectional nationwide survey on EA. We can now develop shared guidelines and provide more reliable prognostic expectations for our patients.

Application of molecular technologies for phosphoproteomic analysis of clinical samples.

The integration of small kinase inhibitors and monoclonal antibodies into oncological practice has opened a new paradigm for treating cancer patients. As proteins are the direct targets of the new generations of targeted therapeutics, many of which are kinase/enzymatic inhibitors, there is an increasing interest in developing technologies capable of monitoring post-translational changes of the human proteome for the identification of new predictive, prognostic and therapeutic biomarkers. It is well known that the vast majority of the activation/deactivation of these drug targets is driven by phosphorylation. This review provides a description of the main proteomic platforms (planar and bead array, reverse phase protein microarray, phosphoflow, AQUA and mass spectrometry) that have successfully been used for measuring changes in phosphorylation level of drug targets and downstream substrates using clinical specimens. Major emphasis was given to the strengths and weaknesses of the different platforms and to the major barriers that are associated with the analysis of the phosphoproteome. Finally, a number of examples of application of the above-mentioned technologies in the clinical setting are reported.

Pilot phase I/II personalized therapy trial for metastatic colorectal cancer: evaluating the feasibility of protein pathway activation mapping for stratifying patients to therapy with imatinib and panitumumab.

This nonrandomized phase I/II trial assessed the efficacy/tolerability of imatinib plus panitumumab in patients affected by metastatic colorectal cancer (mCRC) after stratification to treatment by selection of activated imatinib drug targets using reverse-phase protein array (RPPA). mCRC patients presenting with a biopsiable liver metastasis were enrolled. Allocation to the experimental and control arms was established using functional pathway activation mapping of c-Kit, PDGFR, and c-Abl phosphorylation by RPPA. The experimental arm received run-in escalation therapy with imatinib followed by panitumumab. The control arm received panitumumab alone. Seven patients were enrolled in the study. For three of the seven patients, sequential pre- and post-treatment biopsies were used to evaluate the effect of the therapeutic compounds on the drug targets and substrates. A decrease in the activation level of the drug targets and downstream substrates was observed in two of three patients. Combination therapy increased the activation of the AKT-mTOR pathway and several receptor tyrosine kinases. This study proposes a novel methodology for stratifying patients to personalized treatment based on the activation level of the drug targets. This workflow provides the ability to monitor changes in the signaling pathways after the administration of targeted therapies and to identify compensatory mechanisms.

Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis.

Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

A rare case of simple syndactyly in a puppy.

A case of non-syndromic, complete syndactyly involving all four limbs is described in a three-month-old male crossbreed dog for the first time. Syndactyly is a rare condition in most animal species, in dogs it has been infrequently reported. Findings of clinical, radiographic and cytogenetic analyses are described and demonstrate probably for the first time that numerical and structural chromosome aberrations are not involved in the pathogenesis of this case of syndactyly.

A candidate anti-HIV reservoir compound, auranofin, exerts a selective 'anti-memory' effect by exploiting the baseline oxidative status of lymphocytes.

Central memory (T(CM)) and transitional memory (T(TM)) CD4(+) T cells are known to be the major cellular reservoirs for HIV, as these cells can harbor a transcriptionally silent form of viral DNA that is not targeted by either the immune system or current antiretroviral drug regimens. In the present study, we explored the molecular bases of the anti-HIV reservoir effects of auranofin (AF), a pro-oxidant gold-based drug and a candidate compound for a cure of AIDS. We here show that T(CM) and T(TM) lymphocytes have lower baseline antioxidant defenses as compared with their naive counterpart. These differences are mirrored by the effects exerted by AF on T-lymphocytes: AF was able to exert a pro-differentiating and pro-apoptotic effect, which was more pronounced in the memory subsets. AF induced an early activation of the p38 mitogen-activated protein kinase (p38 MAPK) followed by mitochondrial depolarization and a final burst in intracellular peroxides. The pro-differentiating effect was characterized by a downregulation of the CD27 marker expression. Interestingly, AF-induced apoptosis was inhibited by pyruvate, a well-known peroxide scavenger, but pyruvate did not inhibit the pro-differentiating effect of AF, indicating that the pro-apoptotic and pro-differentiating effects involve different pathways. In conclusion, our results demonstrate that AF selectively targets the T(CM)/T(TM) lymphocyte subsets, which encompass the HIV reservoir, by affecting redox-sensitive cell death pathways.

Ultrasound measurement of reproductive organs in juvenile European sea bass Dicentrarchus labrax.

In this study, ultrasonographic examination was performed thrice, 15 days apart, on juvenile European sea bass Dicentrarchus labrax, from 330 to 360 days of age, to assess the size and the morphology of male and female. Results have proved this method as a suitable and non-invasive procedure to assess sexual differentiation.

Keggin heteropolyacid H3PW12O40 supported on different oxides for catalytic and catalytic photo-assisted propene hydration.

Catalytic and catalytic photo-assisted hydration of propene to form 2-propanol in gas-solid regime at atmospheric pressure and 85 °C were carried out by using a heteropolyacid (POM) supported on different oxides. Binary materials were prepared by impregnation of H3PW12O40 on different commercial and home prepared supports (TiO2, SiO2, WO3, ZrO2, ZnO, Al2O3). Some of the composites were active both for catalytic and catalytic photo-assisted reactions. The Keggin type POM was completely and partially degraded, when supported on ZnO and Al2O3, respectively, and these binary solids always resulted as inactive for both catalytic and catalytic photo-assisted reactions. The supported Keggin POM species played a key role both for the catalytic and the photo-assisted catalytic reactions, due to their strong acidity and ability to form strong oxidant species under UV irradiation, respectively. The contemporary presence of heat and UV light improved the activity of almost all POM supported materials. All materials were characterized by X-ray diffraction (XRD), scanning electron microscopy observations (SEM), diffuse reflectance spectroscopy (DRS), determination of the conduction and valence band energy by photovoltage measurements, Fourier transform infrared spectroscopy (FTIR), NH3-TPD experiments and time resolved microwave conductivity (TRMC).

Individualized therapy for metastatic colorectal cancer.

Systemic therapeutic efficacy is central to determining the outcome of patients with metastatic colorectal cancer (CRC). In these patients, there is a critical need for predictive biomarkers to optimize efficacy whilst minimizing toxicity. The integration of a new generation of molecularly targeted drugs into the treatment of CRC, coupled with the development of sophisticated technologies for individual tumours as well as patient molecular profiling, underlines the potential for personalized medicine. In this review, we focus on the latest progress made within the genomic and proteomic fields, concerning predictive biomarkers for individualized therapy in metastatic CRC.

Detecting population structure and recent demographic history in endangered livestock breeds: the case of the Italian autochthonous donkeys.

Since its domestication, about 5000 years ago, the donkey (Equus asinus) has been extensively used as a work or draft animal in agricultural activities and for the transportation of people and goods. In the last century, technology improvement and growing mechanization strongly affected agriculture and the management and use of this livestock species in the industrialized countries. Nowadays, the use of donkeys for work or transport has almost disappeared, together with the need for mules or hinny breeding. During the last five decades, Italian autochthonous donkey populations suffered from a severe reduction in population size, which led to the extinction of several breeds. At present, eight breeds remain, all classified by FAO as critically endangered or endangered: Asinara, Pantesco, Grigio Siciliano, Romagnolo, Amiatino, Sardo Grigio, Martina Franca, and Ragusano. To evaluate the extant genetic variability of Italian donkeys, we typed 16 microsatellite loci in 258 individuals from these breeds. The results highlighted moderate levels of inbreeding ( F (IS) = 0.127) and a significant partition of genetic variation into breeds, as suggested by fixation index ( F (ST) = 0.109) and analysis of molecular variance (10.86% of total variation assigned to the between-breeds level) analyses. This was confirmed by a Bayesian clustering procedure that also highlighted a further partitioning at lower hierarchical levels corresponding to the farms of origin. This evidence suggests that an effective management strategy for Italian donkey populations should focus on breeds as conservation units. However, this requires a synergic management strategy at the farm level to maintain diversity and avoid inbreeding.

AMPK inhibition enhances apoptosis in MLL-rearranged pediatric B-acute lymphoblastic leukemia cells.

The serine/threonine kinase AMP-activated protein kinase (AMPK) and its downstream effectors, including endothelial nitric oxide synthase and BCL-2, are hyperactivated in B-cell precursor-acute lymphoblastic leukemia (BCP-ALL) cells with MLL gene rearrangements. We investigated the role of activated AMPK in supporting leukemic cell survival and evaluated AMPK as a potential drug target. Exposure of leukemic cells to the commercial AMPK inhibitor compound C resulted in massive apoptosis only in cells with MLL gene rearrangements. These results were confirmed by targeting AMPK with specific short hairpin RNAs. Compound C-induced apoptosis was associated with mitochondrial membrane depolarization, reactive oxygen species production, cytochrome c release and caspases cleavage, indicating intrinsic apoptosis pathway activation. Treatment with low concentrations of compound C resulted in a strong antileukemic activity, together with cytochrome c release and cleavage of caspases and poly(ADP-ribose) polymerase, also in MLL-rearranged primary BCP-ALL samples. Moreover, AMPK inhibition in MLL-rearranged cell lines synergistically enhanced the antiproliferative effects of vincristine, daunorubicin, cytarabine, dexamethasone and L-asparaginase in most of the evaluated conditions. Taken together, these results indicate that the activation of the AMPK pathway directly contributes to the survival of MLL-rearranged BCP-ALL cells and AMPK inhibitors could represent a new therapeutic strategy for this high-risk leukemia.

Homologous control of protein signaling networks.

In a previous paper we introduced a method called augmented sparse reconstruction (ASR) that identifies links among nodes of ordinary differential equation networks, given a small set of observed trajectories with various initial conditions. The main purpose of that technique was to reconstruct intracellular protein signaling networks. In this paper we show that a recursive augmented sparse reconstruction generates artificial networks that are homologous to a large, reference network, in the sense that kinase inhibition of several reactions in the network alters the trajectories of a sizable number of proteins in comparable ways for reference and reconstructed networks. We show this result using a large in-silico model of the epidermal growth factor receptor (EGF-R) driven signaling cascade to generate the data used in the reconstruction algorithm. The most significant consequence of this observed homology is that a nearly optimal combinatorial dosage of kinase inhibitors can be inferred, for many nodes, from the reconstructed network, a result potentially useful for a variety of applications in personalized medicine.

Platform for establishing interlaboratory reproducibility of selected reaction monitoring-based mass spectrometry peptide assays.

Mass spectrometry (MS) is an attractive alternative to quantification of proteins by immunoassays, particularly for protein biomarkers of clinical relevance. Reliable quantification requires that the MS-based assays are robust, selective, and reproducible. Thus, the development of standardized protocols is essential to introduce MS into clinical research laboratories. The aim of this study was to establish a complete workflow for assessing the transferability and reproducibility of selected reaction monitoring (SRM) assays between clinical research laboratories. Four independent laboratories in North America, using identical triple-quadrupole mass spectrometers (Quantum Ultra, Thermo), were provided with standard protocols and instrumentation settings to analyze unknown samples and internal standards in a digested plasma matrix to quantify 51 peptides from 39 human proteins using a multiplexed SRM assay. The interlaboratory coefficient of variation (CV) was less than 10% for 25 of 39 peptides quantified (12 peptides were not quantified based upon hydrophobicity) and exhibited CVs less than 20% for the remaining peptides. In this report, we demonstrate that previously developed research platforms for SRM assays can be improved and optimized for deployment in clinical research environments.

Genetic heterogeneity and selection signature at the KIT gene in pigs showing different coat colours and patterns.

Mutations in the porcine KIT gene (Dominant white locus) have been shown to affect coat colours and colour distribution in pigs. We analysed this gene in several pig breeds and populations (Sicilian black, completely black or with white patches; Cinta Senese; grey local population; Large White; Duroc; Hampshire; Pietrain; wild boar; Meishan) with different coat colours and patterns, genotyping a few polymorphisms. The 21 exons and parts of the intronic regions were sequenced in these pigs and 69 polymorphisms were identified. The grey-roan coat colour observed in a local grey population was completely associated with a 4-bp deletion of intron 18 in a single copy KIT gene, providing evidence that this mutation characterizes the I(d) allele described in the early genetic literature. The white patches observed in black Sicilian pigs were not completely associated with the presence of a duplicated KIT allele (I(p) ), suggesting that genetic heterogeneity is a possible cause of different coat colours in this breed. Selection signature was evident at the KIT gene in two different belted pig breeds, Hampshire and Cinta Senese. The same mutation(s) may cause the belted phenotype in these breeds that originated in the 18th-19th centuries from English pigs (Hampshire) and in Tuscany (Italy) in the 14th century (Cinta Senese). Phylogenetic relationships of 28 inferred KIT haplotypes indicated two clades: one of Asian origin that included Meishan and a few Sicilian black haplotypes and another of European origin.

Organic management of dietary rosemary extract in dairy sheep: effects on milk quality and clotting properties.

The increasing demand for animals from organic breeding systems has increased interest in certain natural substances, called nutraceuticals, to stimulate the organic defenses of the animals. The aim of this trial was to study the effects of dietary rosemary extract in 36 ewes, from 57 to 154 days of lactation, divided into three groups: CTR (basal diet), ROXLD (600 mg extract/head/day) and ROXHD (1,200 mg extract/head/day). A significantly higher quantity of milk and quantitative daily production of protein, casein, fat, and lactose were observed in the milk of animals in the ROXHD group compared with milk from animals in the CTR and ROXLD groups. No significant differences were observed for somatic cell counts, considering that treated groups showed lower values compared with controls. A significant decrease in clotting time (r) and increase in curd firmness (a ( 30 )) were observed in milk of both treated groups (ROXLD and ROXHD) compared with the CTR group. These results could be related to the significantly higher acidity values, pH and SH degrees , observed in the milk of animals from the treated groups. Dietary rosemary extract in dairy sheep enhanced milk yield, quality, and renneting properties due to its "natural, functional ingredients."

Nanoparticle technology: addressing the fundamental roadblocks to protein biomarker discovery.

Clinically relevant biomarkers exist in blood and body fluids in extremely low concentrations, are masked by high abundance high molecular weight proteins, and often undergo degradation during collection and transport due to endogenous and exogenous proteinases. Nanoparticles composed of a N-isopropylacrylamide hydrogel core shell functionalized with internal affinity baits are a new technology that can address all of these critical analytical challenges for disease biomarker discovery and measurement. Core-shell, bait containing, nanoparticles can perform four functions in one step, in solution, in complex biologic fluids (e.g. blood or urine): a) molecular size sieving, b) complete exclusion of high abundance unwanted proteins, c) target analyte affinity sequestration, and d) complete protection of captured analytes from degradation. Targeted classes of protein analytes sequestered by the particles can be concentrated in small volumes to effectively amplify (up to 100 fold or greater depending on the starting sample volume) the sensitivity of mass spectrometry, western blotting, and immunoassays. The materials utilized for the manufacture of the particles are economical, stable overtime, and remain fully soluble in body fluids to achieve virtually 100 percent capture of all solution phase target proteins within a few minutes.