Enhancing the electron transfer capacity and subsequent color removal in bioreactors by applying thermophilic anaerobic treatment and redox mediators

The effect of temperature, hydraulic retention time (HRT) and the redox mediator anthraquinone-2,6-disulfonate (AQDS), on electron transfer and subsequent color removal from textile wastewater was assessed in mesophilic and thermophilic anaerobic bioreactors. The results clearly show that compared with mesophilic anaerobic treatment, thermophilic treatment at 55 degrees C is an effective approach for increasing the electron transfer capacity in bioreactors, and thus improving the decolorization rates. Furthermore, similar color removals were found at 55 degrees C between the AQDS-free and AQDS-supplemented reactors, whereas a significant difference (up to 3.6-fold) on decolorization rates occurred at 30 degrees C. For instance, at an HRT of 2.5 h and in the absence of AQDS, the color removal was 5.3-fold higher at 55 degrees C compared with 30 degrees C. The impact of a mix of mediators with different redox potentials on the decolorization rate was investigated with both industrial textile wastewater and the azo dye Reactive Red 2 (RR2). Color removal of RR2 in the presence of anthraquinone-2-sulfonate (AQS) (standard redox potential E(0)' of -225 mV) was 3.8-fold and 2.3-fold higher at 30 degrees C and 55 degrees C, respectively, than the values found in the absence of AQS. Furthermore, when the mediators 1,4-benzoquinone (BQ) (E(0)' of +280 mV), and AQS were incubated together, there was no improvement on the decolorization rates compared with the bottles solely supplemented with AQS. Results imply that the use of mixed redox mediators with positive and negative E(0)' under anaerobic conditions is not an efficient approach to improve color removal in textile wastewaters.     

EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival,Cell Invasion,Focal Adhesions,and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130^Cas. Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130^Cas. WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.     

Telomerase Recruitment in Saccharomyces cerevisiae Is Not Dependent on Tel1-Mediated Phosphorylation of Cdc13

In Saccharomyces cerevisiae, association between the Est1 telomerase subunit and the telomere-binding protein Cdc13 is essential for telomerase to be recruited to its site of action. A current model proposes that Tel1 binding to telomeres marks them for elongation, as the result of phosphorylation of a proposed S/TQ cluster in the telomerase recruitment domain of Cdc13. However, three observations presented here argue against one key aspect of this model. First, the pattern of Cdc13 phosphatase-sensitive isoforms is not altered by loss of Tel1 function or by mutations introduced into two conserved serines (S249 and S255) in the Cdc13 recruitment domain. Second, an interaction between Cdc13 and Est1, as monitored by a two-hybrid assay, is dependent on S255 but Tel1-independent. Finally, a derivative of Cdc13, cdc13–(S/TQ)₁₁→(S/TA)₁₁, in which every potential consensus phosphorylation site for Tel1 has been eliminated, confers nearly wild-type telomere length. These results are inconsistent with a model in which the Cdc13–Est1 interaction is regulated by Tel1-mediated phosphorylation of the Cdc13 telomerase recruitment domain. We propose an alternative model for the role of Tel1 in telomere homeostasis, which is based on the assumption that Tel1 performs the same molecular task at double-strand breaks (DSBs) and chromosome termini.TELOMERE length homeostasis is a highly regulated process that must balance telomere loss (as the result of incomplete replication and/or nucleolytic degradation) with telomeric repeat addition (through the action of telomerase and/or recombination). In the budding yeast Saccharomyces cerevisiae, a key regulatory event is recruitment of telomerase to chromosome ends by the telomere end-binding protein Cdc13 (Nugent et al. 1996; Evans and Lundblad 1999; Pennock et al. 2001; Bianchi et al. 2004; Chan et al. 2008). Recruitment relies on a direct interaction between Cdc13 and the Est1 subunit of telomerase (Pennock et al. 2001), which brings the catalytic core of the enzyme to its site of action. Disruption of this interaction, due to mutations in either CDC13 (cdc13-2) or EST1 (est1-60), results in an Est (ever-shorter-telomere) phenotype, as manifested by progressive telomere shortening and an eventual senescence phenotype. The recruitment activity of Cdc13, which resides in a 15-kDa N-terminal domain (Pennock et al. 2001), is sufficient to direct telomerase even to nontelomeric sites (Bianchi et al. 2004). As predicted by the recruitment model, association of telomerase with telomeres is greatly reduced in strains expressing the recruitment-defective cdc13-2 allele (Chan et al. 2008).Telomerase action at individual telomeres is highly regulated. Using an assay that monitors telomere addition at single nucleotide resolution (single telomere extension, STEX), Lingner and colleagues showed that only ∼7% of telomeres with wild-type (i.e., 300 bp) length are elongated by telomerase during a single cell cycle (Teixeira et al. 2004). However, as telomere length declines, the extension frequency increases: ∼20% of telomeres 200 bp in length and >40% of 100-bp-long telomeres are elongated (Teixeira et al. 2004; Arneric and Lingner 2007). The mechanism by which telomerase distinguishes short from long telomeres has been the subject of intense investigation. Work from a number of laboratories has led to the proposal that Tel1-dependent phosphorylation of Cdc13 at underelongated telomeres mediates the interaction between Cdc13 and the telomerase-associated Est1 protein, thus ensuring that telomerase is directed to the shortest telomeres in a population. In support of this model, the Est1 and Est2 telomerase subunits exhibit enhanced association with telomeres that have been artificially shortened, whereas Cdc13 displays length-independent association with telomeres (Bianchi and Shore 2007; Sabourin et al. 2007). This suggests that the preferential elongation of shorter telomeres is controlled at the level of recruitment of the telomerase holoenzyme by Cdc13. Furthermore, efficient association of Est1 and Est2 with chromosome ends requires Tel1 and Mre11 (which acts in the same pathway as Tel1 for telomere length regulation; Nugent et al. 1998; Ritchie and Petes 2000) but not Mec1 (Takata et al. 2005; Goudsouzian et al. 2006). Tel1 itself is also telomere bound (Takata et al. 2004), with enhanced binding to shorter telomeres (Bianchi and Shore 2007; Hector et al. 2007; Sabourin et al. 2007; Abdallah et al. 2009), although there is considerable controversy over the degree and timing of Tel1 association with chromosome termini during the cell cycle. As expected for a key regulator of the interaction between Cdc13 and a telomerase subunit, a tel1-Δ strain has short telomeres (Lustig and Petes 1986), although telomere length is not impaired enough to confer the Est phenotype displayed by cdc13-2 and est1-60 strains.Implicit in the above proposal is that Cdc13 must be a direct substrate of Tel1. In support of this, Teng and colleagues reported several years ago that the recruitment domain of Cdc13 has a cluster of potential Tel1 (and/or Mec1) phosphorylation sites (Tseng et al. 2006). Substrates of the DNA damage kinases often contain several closely spaced phosphorylation sites, termed S/TQ cluster domains (SCDs), which are considered a structural hallmark of many DNA damage-response proteins (Traven and Heierhorst 2005). On the basis of in vitro kinase assays with GST fusions to 75- to 90-amino-acid portions of the Cdc13 recruitment domain, Tseng et al. 2006 concluded that four SQ sites in the recruitment domain of Cdc13 are overlapping substrates for both Tel1 and Mec1, leading to the proposal that telomerase recruitment in S. cerevisiae is regulated by Tel1-dependent phosphorylation of Cdc13.The above model makes a key prediction: in a tel1-Δ strain, telomerase should no longer exhibit a length-dependent pattern of elongation. However, preferential elongation of short telomeres still occurs at native chromosome ends in the absence of Tel1 (Arneric and Lingner 2007). In addition, Petes and colleagues have argued, on the basis of epistasis data, that Tel1 performs an indirect role in the telomerase pathway, rather than directly targeting a telomerase regulator (Ritchie et al. 1999; Ritchie and Petes 2000). These observations are not easily explained, if preferential recognition of short telomeres by telomerase is mediated by Tel1-dependent phosphorylation of Cdc13. In this current study, we have re-examined the evidence for phosphorylation of Cdc13 as a regulatory mechanism for telomere length homeostasis. We report on a series of observations that indicate that Tel1 contributes to telomere length control through a mechanism other than phosphorylation of the Cdc13 S/TQ cluster.     

Membrane topology of the chromate transporter ChrA of Pseudomonas aeruginosa

The membrane topology of the plasmid-encoded Pseudomonas aeruginosa ChrA protein, which effluxes chromate ions, was determined by the analysis of translational fusions with reporter enzymes alkaline phosphatase and beta-galactosidase. A novel 13-TMS (transmembrane segments) topology, with the N-terminus located in the cytoplasm and the C-terminus in the periplasmic space, was consistent with the enzyme activities determined in both Escherichia coli and P. aeruginosa. Alignment of the two halves of ChrA showed significant sequence homology, with TMS I, II, III, IV, V and VI displaying similarity to TMS VIII, IX, X, XI, XII and XIII, respectively, although with opposite membrane orientations. This suggests that ChrA arose from the duplication of a gene encoding a 6-TMS ancestral protein, followed by the insertion of extra TMS VII. These data also suggest that the two halves of ChrA may carry out distinct functions for the transport of chromate.     

Dehydroepiandrosterone decreases while cortisol increases in vitro growth and viability of Entamoeba histolytica

In vitro exposure of Entamoeba histolytica trophozoites to the sex steroids 17beta-estradiol, progesterone, and dehydrotestosterone had little effect on parasite viability or proliferation. However, treatment with the adrenal steroid dehydroepiandrosterone (DHEA) markedly inhibited parasite proliferation, adherence and motility, and at a certain dose it induced trophozoite lysis. The opposite effect on proliferation was found when the trophozoites were exposed to cortisol. Moreover, DHEA decreased while cortisol increased the parasite's DNA synthesis determined by 3H-thymidine incorporation. Trophozoite lysis by DHEA appeared to be caused by a necrotic rather than an apoptotic process, as observed in propidium iodide and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assays. A possible mechanisms of action was derived from experiments demonstrating that the activity of a putative 3-hydroxy-3-methyl glutaryl CoA reductase detected in trophozoite extracts was inhibited in the presence of DHEA. Contrary to its in vitro inhibitory effect, in vivo administration of DHEA to infected hamsters resulted in exacerbation of the amebic liver abscesses. These results demonstrated that androgen steroids act directly upon E. histolytica growth and viability, and may shed new light on some age and gender differences in disease progression, as well as finding application in the drug treatment of human amebiasis.     

Design and experimental evaluation of adjustable bone plates for mandibular fracture fixation

Conventional bone plates are commonly used for surgical mandibular fracture fixation. Improper alignment between bone segments, however, can result in malocclusion. Current methods of fixation require a surgeon to visually align segments of bone and affix a metal plate using bone screws, after which little can be done to adjust alignment. A method of adjusting fracture alignment after plate placement, without screw removal, presents an improvement over costly and risky revision surgery. A modified bone plate has been designed with a deformable section to give surgeons the ability to reduce misalignments at the fracture site. The mechanics of deformation for various adjustment mechanisms was explored analytically, numerically, and experimentally to ensure that the adjustable plate is comparable to conventional bone plates. A static force of 358.8 N is required to deform the adjustable bone plate, compared with predicted values of 351 N using numerical simulation and 362 N using a simple beam theory. Dynamic testing was performed to simulate in vivo loading conditions and evaluate load-capacity in both deformed and un-deformed bone plates. Results indicate that bending stiffness of a rectangular bone plate is 709 N/mm, compared with 174 N/mm for an octagonal plate and 176 N/mm for standard plates. Once deformed, the rectangular and octagonal plates had a stiffness of 323 N/mm and 228 N/mm, respectively. Un-deformed and deformed adjustable bone plates have efficacy in bone segment fixation and healing.     

Foliar application of urea to “Sauvignon Blanc” and “Merlot” vines: doses and time of application

A careful control of the N nutritional status of grapevines can have a determining effect on wine characteristics; therefore a suitable management of N fertilization might allow some wine parameters to be modified, thereby improving product quality. The aim of this study was to determine the effect of foliar application of urea at different doses and different times of the growing season on the parameters of Sauvignon Blanc and Merlot grape juice. The research described herein involved Sauvignon Blanc and Merlot grapevines (V. vinifera L.) at a commercial vineyard and was conducted over 2 years. In the first year, N treatment involved a foliar application at a dose of 10 kg N ha^?1 during veraison, whereas in the second year it involved a foliar urea application at two doses (10 and 50 kg N ha^?1) and at three different times—3 weeks before veraison, during veraison and 3 weeks after veraison. In this second year, the urea applied at a dose of 10 kg N ha^?1 was isotopically labelled with 1% ¹⁵N. Chemical parameters, yeast assimilable N, amino acid content, amino acid profile and N isotopic composition were determined for all treatments. Grape and grape-juice parameters for Merlot were found to be more affected by N fertilization than for Sauvignon Blanc and were also more affected during the second year than during the first year, thus indicating that the climatic characteristics of each campaign could affect these parameters. The yeast assimilable N in grape juice was found to be higher for late applications of foliar urea, with application of the higher dose of urea during veraison increasing the amino acid and proline contents in both varieties. The isotopic analysis data showed that the urea applied to leaves was transferred to the berries, with the maximum translocation in Sauvignon Blanc occurring for the post-veraison treatment and in Merlot for the veraison treatment. We can therefore conclude that foliar application of urea could modify grape juice quality and could therefore be used as a tool for obtaining quality wines.     

Dally and Notum regulate the switch between low and high level Hedgehog pathway signalling

During development, secreted morphogens, such as Hedgehog (Hh), control cell fate and proliferation. Precise sensing of morphogen levels and dynamic cellular responses are required for morphogen-directed morphogenesis, yet the molecular mechanisms responsible are poorly understood. Several recent studies have suggested the involvement of a multi-protein Hh reception complex, and have hinted at an understated complexity in Hh sensing at the cell surface. We show here that the expression of the proteoglycan Dally in Hh-receiving cells in Drosophila is necessary for high but not low level pathway activity, independent of its requirement in Hh-producing cells. We demonstrate that Dally is necessary to sequester Hh at the cell surface and to promote Hh internalisation with its receptor. This internalisation depends on both the activity of the hydrolase Notum and the glycosyl-phosphatidyl-inositol (GPI) moiety of Dally, and indicates a departure from the role of the second glypican Dally-like in Hh signalling. Our data suggest that hydrolysis of the Dally-GPI by Notum provides a switch from low to high level signalling by promoting internalisation of the Hh-Patched ligand-receptor complex.     

The ChrA homologue from a sulfur-regulated gene cluster in cyanobacterial plasmid pANL confers chromate resistance

The cyanobacterium Synechococcus elongatus strain PCC 7942 possesses pANL, a plasmid rich in genes related to sulfur metabolism. One of these genes, srpC, encodes the SrpC protein, a homologue of the CHR chromate ion transporter superfamily. The srpC gene was cloned and expressed in Escherichia coli and its role in relation to sulfate and chromate was analyzed. srpC was unable to complement the growth of an E. coli cysA sulfate uptake mutant when sulfate was utilized as a sole sulfur source, suggesting that SrpC is not a sulfate transporter. Expression of srpC in E. coli conferred chromate resistance and caused diminished chromate uptake. These results suggest that the S. elongatus SrpC protein functions as a transporter that extrudes chromate ions from the cell’s cytoplasm, and further demonstrate the close relationship between sulfate and chromate metabolism in this organism.     

Simultaneous oxidation of ammonium and p-cresol linked to nitrite reduction by denitrifying sludge

The metabolic capability of denitrifying sludge to oxidize ammonium and p-cresol was evaluated in batch cultures. Ammonium oxidation was studied in presence of nitrite and/or p-cresol by 55 h. At 50 mg/L NH₄⁺-N and 76 mg/L NO₂⁻-N, the substrates were consumed at 100% and 95%, respectively, being N₂ the product. At 50 mg/L NH₄⁺-N and 133 mg/L NO₂⁻-N, the consumption efficiencies decreased to 96% and 70%, respectively. The increase in nitrite concentration affected the ammonium oxidation rate. Nonetheless, the N₂ production rate did not change. In organotrophic denitrification, the p-cresol oxidation rate was slower than ammonium oxidation. In litho-organotrophic cultures, the p-cresol and ammonium oxidation rates were affected at 133 mg/L NO₂⁻-N. Nonetheless, at 76 mg/L NO₂⁻-N the denitrifying sludge oxidized ammonium and p-cresol, but at different rate. Finally, this is the first work reporting the simultaneous oxidation of ammonium and p-cresol with the production of N₂ from denitrifying sludge.