Optimization of P compounds recovery from aerobic sludge by chemical modeling and response surface methodology combination.

Phosphorus recovery has drawn much attention during recent years, due to estimated limited available quantities, and to the harmful environmental impact that it may have when freely released into aquatic environments. Struvite precipitation from wastewater or biological sludge is among the preferred approaches applied for phosphorus recovery, as it results in the availability of valuable fertilizer materials. This process is mostly affected by pH and presence of competitive ions in solution. Modeling and optimization of the precipitation process may help understanding the optimal conditions under which the most efficient recovery could be achieved. In this study, a combination of chemical equilibrium modeling and response surface methodology (RSM) was applied to this aim to aerobic sludge from a plant in Italy. The results identify optimum chemical parameters values for best phosphorus precipitation recovery and removal efficiencies, respectively. Identification of optimal conditions for process control is of great importance for implementing pilot scale struvite precipitation and achieve efficient phosphorus recovery.

Formation of Hexamethylenetetramine (HMT) from HCHO and NH3--Relevance to Prebiotic Chemistry and B3LYP Consideration.

Despite its importance in the prebiotic and biochemical fields, a complete theoretical study of the formation of hexamethylenetetramine (HMT) starting from its precursors ammonia and formaldehyde has not received due considerations in the literature with regard to the thermodynamic feasibility of many of the mechanistically proposed intermediates in its formation. Most of the studies in this area have been mostly concerned with the initial steps of the reaction between formaldehyde and ammonia, while poor attention is dedicated to successive steps. In this article, different results from published literature were critically considered and the most probable hypothesis regarding the mechanism of HMT formation is discussed on the basis of B3LYP calculations of free energies.

Exploring the molecular and chemical-physical aspects of low-dose irradiation using radio-tolerant plant cells.

Animals and plants show different levels of radio-sensitivity, with safe dose values in the 0.001-1 and 1-100 Gy range, respectively. The increased radio-tolerance observed in plant cells might represent a valuable tool to investigate the events underlying the low dose (LD) response in the highly radio-sensitive animal cells. The use of radio-resistant plant systems would allow investigation of the LD effects using irradiation conditions that can be easily managed, without the technical constrains currently encountered with animal systems. The basal knowledge on the molecular mechanisms involved in the DNA damage response in plants is rapidly expanding, revealing common features with animal cells at the level of DNA damage sensing/repair, transduction pathways and antioxidant response. The present work provides a short update of the current literature dealing with the DNA damage response in animal and plant cells exposed to LD treatments (ionising radiation, particularly gamma ray) combined with the more recent advances in free radical research.

Sunlight-promoted photocatalytic hydrogen gas evolution from water-suspended cellulose: a systematic study.

This work presents a systematic study of cellulose (CLS) as a sacrificial biomass for photocatalytic H2 evolution from water. The idea is indeed to couple a largely available and not expensive biomass, and water, with a renewable energy like solar radiation. An aqueous CLS suspension irradiated either at 366 nm (UV-A) or under sunlight in the presence of Pt/TiO2 behaves as a H2 evolving system. The effects of irradiation time, catalyst and CLS concentrations, pH and water salinity are studied. Addition of CLS to the sample significantly improved H2 evolution from water splitting, with yields up to ten fold higher than those observed in neat water. The mechanism of the photocatalytic process relies on the TiO2-mediated CLS hydrolysis, under irradiation. The polysaccharide depolymerisation generates water-soluble species and intermediates, among them 5-hydroxymethylfurfural (HMF) was identified. These intermediates are readily oxidized following the glucose photoreforming, thus enhancing water hydrogen ion reduction to give gas-phase H2. The formation of "colored" by-products from HMF self-polymerization involves a sort of "in situ dye sensitization" that allows an effective photoreaction even under solar light. The procedure is evaluated and successfully extended on cellulosic biomasses, i.e. rice husk and alfalfa (Medicago sativa) stems, not previously investigated for this application.

Synergistic exposure of rice seeds to different doses of γ-ray and salinity stress resulted in increased antioxidant enzyme activities and gene-specific modulation of TC-NER pathway.

Recent reports have underlined the potential of gamma (γ)-rays as tools for seed priming, a process used in seed industry to increase seed vigor and to enhance plant tolerance to biotic/abiotic stresses. However, the impact of γ -rays on key aspects of plant metabolism still needs to be carefully evaluated. In the present study, rice seeds were challenged with different doses of γ -rays and grown in absence/presence of NaCl to assess the impact of these treatments on the early stages of plant life. Enhanced germination efficiency associated with increase in radicle and hypocotyl length was observed, while at later stages no increase in plant tolerance to salinity stress was evident. APX, CAT, and GR were enhanced at transcriptional level and in terms of enzyme activity, indicating the activation of antioxidant defence. The profiles of DNA damage accumulation were obtained using SCGE and the implication of TC-NER pathway in DNA damage sensing and repair mechanisms is discussed. OsXPB2, OsXPD, OsTFIIS, and OsTFIIS-like genes showed differential modulation in seedlings and plantlets in response to γ -irradiation and salinity stress. Altogether, the synergistic exposure to γ -rays and NaCl resulted in enhanced oxidative stress and proper activation of antioxidant mechanisms, thus being compatible with plant survival.

Single Cell Gel Electrophoresis (Comet) assay with plants: research on DNA repair and ecogenotoxicity testing.

Single Cell Gel Electrophoresis is currently used to investigate the cell response to genotoxic agents as well as to several biotic and abiotic stresses that lead to oxidative DNA damage. Different versions of Single Cell Gel Electrophoresis have been developed in order to expand the range of DNA lesions that can be detected and guidelines for their use in genetic toxicology have been provided. Applications of Single Cell Gel Electrophoresis in plants are still limited, compared to animal systems. This technique is now emerging as a useful tool in assessing the potential of higher plants as stable sensors in ecosystems and source of information on the genotoxic impact of dangerous pollutants. Another interesting application of Single Cell Gel Electrophoresis deals with Mutation Breeding or the combined use of irradiation and in vitro culture technique to enhance genetic variability in elite plant genotypes. SCGE, in combination with in situ detection of Reactive Oxygen Species (ROS) induced by γ-rays and expression analysis of both DNA repair and antioxidant genes, can be used to gather information on the radiosensitivity level of the target plant genotypes.

RNA-Seq analysis discloses early senescence and nucleolar dysfunction triggered by Tdp1α depletion in Medicago truncatula.

An intron-spliced hairpin RNA approach was used for the targeted silencing of the MtTdp1α gene encoding the αisoform of tyrosyl-DNA phosphodiesterase 1 in Medicago truncatula Gaertn. Tyrosyl-DNA phosphodiesterase 1, involved in the repair of DNA topoisomerase I-mediated DNA damage, has been poorly investigated in plants. RNA-Seq analysis, carried out in the MtTdp1α-depleted plants, revealed different levels of transcriptional modulation (up- and down-regulation, alternative splicing, activation of alternative promoter) in genes involved in DNA damage sensing, DNA repair, and chromatin remodelling. It is suggested that the MtTdp1α gene has new, previously undetected roles in maintaining genome integrity. Up-regulation of senescence-associated genes and telomere shortening were observed. Moreover, impaired ribosome biogenesis indicated that the MtTdp1α gene is required for the nucleolar function. In agreement with the RNA-Seq data, transmission electron microscopy detected an altered nucleolar architecture in the MtTdp1α-depleted cells. Based on the reported data, a working hypothesis related to the occurrence of a nucleolar checkpoint in plant cells is proposed.

Understanding the molecular pathways associated with seed vigor.

Farmers and growers are constantly looking for high quality seeds able to ensure uniform field establishment and increased production. Seed priming is used to induce pre-germinative metabolism and then enhance germination efficiency and crop yields. It has been hypothesized that priming treatments might also improve stress tolerance in germinating seeds, leaving a sort of 'stress memory'. However, the molecular bases of priming still need to be clarified and the identification of molecular indicators of seed vigor is nowadays a relevant goal for the basic and applied research in seed biology. It is generally acknowledged that enhanced seed vigor and successful priming depend on DNA repair mechanisms, activated during imbibition. The complexity of the networks of DNA damage control/repair functions has been only partially elucidated in plants and the specific literature that address seeds remains scanty. The DNA repair pathways hereby described (Nucleotide and Base Excision Repair, Non-Homologous End Joining, Homologous Recombination) play specific roles, all of them being critical to ensure genome stability. This review also focuses on some novel regulatory mechanisms of DNA repair (chromatin remodeling and small RNAs) while the possible use of telomere sequences as markers of aging in seed banks is discussed. The significant contribution provided by Electron Paramagnetic Resonance in elucidating the kinetics of seed aging, in terms of free radical profiles and membrane integrity is reported.

The TFIIS and TFIIS-like genes from Medicago truncatula are involved in oxidative stress response.

The cDNA sequence coding for a novel putative TFIIS (transcription elongation factor II-S), hereby named MtTFIIS-like, was isolated from barrel medic (Medicago truncatula Gaertn.) by reverse transcriptase-polymerase chain reaction. The nucleotide sequence contains an open reading frame of 1074 bp, predicting a 40.0 kDa protein, conserved among plant species. The N-terminal region of the MtTFIIS-like protein includes a LW motif, characterized by highly conserved leucine (L) and tryptophan (W) residues, also found in the canonical TFIIS protein, elongin A (transcription elongation factor S-III) and CRSP70 (cofactor required for Sp1 activation), while a proline-rich region is present in the C-terminal domain. The expression profiles of the MtTFIIS-like gene were evaluated by quantitative real-time PCR (QRT-PCR) in barrel medic plantlets grown in vitro under oxidative stress conditions induced by copper (CuCl(2) 0.05, 0.1 and 0.2mM) and polyethylene glycol (PEG6000 50, 100 and 150 g/L), respectively. Both stress agents caused ROS (reactive oxygen species) accumulation. Moreover, EPR spectra of leaves from plantlets exposed to toxic copper doses confirmed that the heavy metal is translocated from roots to the aerial parts, where it is found predominantly in the Cu(2+) redox state. The MtTFIIS-like gene expression was significantly enhanced (up to 2.9-fold) in aerial parts of copper-treated plants, and in roots (up to 4.4-fold) in response to PEG treatments. The expression profiles of the MtTFIIS-like gene were compared to those of the MtTFIIS gene, encoding the canonical TFIIS protein, which was similarly up-regulated in response to both stresses. Interestingly, the MtTFIIS-like and MtTFIIS genes were significantly up-regulated (up to 3.2- and 4.3-fold, respectively) during seed imbibition, a physiological process which requires active DNA repair. Based on the reported data, the possible roles played in planta by the novel MtTFIIS-like gene are discussed.

Effect of cholesterol depletion and temperature on the isolation of detergent-resistant membranes from human erythrocytes.

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C(12)E(8)) at 37 degrees C, and by treatment at 4 degrees C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37 degrees C, or from cholesterol-depleted cells at 4 degrees C, for both detergents. For 5-SASL only, increased S values were measured in 4 degrees C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C(12)E(8) DRMs prepared at 4 degrees C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C(12)E(8) DRMs. However, contrary to the 4 degrees C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C(12)E(8) treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C(12)E(8) to the study of DRMs.

Recognition and sensing of nucleoside monophosphates by a dicopper(II) cryptate.

The dimetallic cryptate [Cu(2)(II)(1)](4+) selectively recognizes guanosine monophosphate with respect to other nucleoside monophosphates (NMPs) in a MeOH/water solution at pH 7. Recognition is efficiently signaled through the displacement of the indicator 6-carboxyfluorescein bound to the receptor, monitoring its yellow fluorescent emission. Titration experiments evidenced the occurrence of several simultaneous equilibria involving 1:1 and 2:1 receptor/NMP and receptor/indicator complexes. It was demonstrated that the added NMP displaces the indicator from the 2:1 receptor/indicator complex, forming the 1:1 receptor/analyte inclusion complex. Recognition selectivity is thus ascribed to the nature of nucleotide donor atoms involved in the coordination and their ability to encompass the Cu(II)-Cu(II) distance within the cryptate.

Resistance of human erythrocyte membranes to Triton X-100 and C12E8.

Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4 degrees C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C12E8. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C12E8, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C12E8 in comparison to intact cells, while the difference in the S values between Triton X-100 and C12E8 DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C12E8 detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs.

The effect of gamma-irradiation on PLGA/PEG microspheres containing ovalbumin.

Poly(ethylene glycol) (PEG) and sodium chloride (NaCl) are excipients used in PLGA microsphere preparation to stabilize proteins and reduce their burst release. No information is till now available in the literature on the effect due to the use of such excipients on the biopharmaceutical performance of gamma-irradiated microparticulate systems. On this purpose, different batches of microspheres containing ovalbumin (OVA) were prepared by using a PLGA 50:50 (average Mr: 13000), different amounts of PEG (Mr: 400 or 4000) and/or sodium chloride. The non-irradiated and irradiated microspheres were characterized in terms of morphology (SEM, particle size distribution), OVA and PEG content and in vitro OVA release. Radiolysis mechanisms of OVA and OVA loaded microspheres were investigated by EPR analysis. Gamma irradiation affects either microsphere morphology or the release of OVA as a function of the amount of PEG, and the use of NaCl. Irradiation significantly reduces release rate of protein from the microspheres containing 15% and 30% of PEG and from controls (microspheres without additives), while no significative effect on protein release rate is highlighted on microspheres containing lower amounts of PEG. EPR investigation shows that increasing amounts of PEG up to 30% have a perturbation effect on OVA radiolysis path.

The influence of solid-state molecular organization on the reaction paths of thiyl radicals.

Electron paramagnetic resonance (EPR) spectroscopy has been employed to investigate the effect of solid-state molecular organization on the reaction of thiyl radicals with thiols. In an irradiated C18H37SH/thiourea clathrate, the conversion of thiyl to perthiyl radicals is substantial, due to the head-to-head arrangement of the reactants within the channels and the suppression of other possible competing reactions due to hindrance by the clathrate walls. The perthiyl radical was identified using EPR analysis of its molecular dynamics within the clathrate channels. Irradiated polyethylene film containing 30% C18H37SH afforded a negligible conversion of thiyl to perthiyl radicals because of the random distribution of reactants. These results suggest that in the absence of favorable structure-control effects, the reaction between RS* and RSH is unimportant with respect to other competing reactions. Perthiyl radicals are also the major product in the vacuum solid-state radiolysis of lysozyme. A proposal of the mechanism involved in all cases is based on the equilibrium RS* + RSH <==> RSS*(H)R, followed by the irreversible conversion of the sulfuranyl radical to the perthiyl radical. As the equilibrium is strongly shifted to the left, the intermediate sulfuranyl radicals were not detected, but the lack of other competing reactions for the thiyl radicals caused the formation of perthiyl radicals to become the major path in the clathrate and in solid lysozyme radiolysis.