Divergent evolution toward sex chromosome-specific gene regulation in Drosophila.

The dosage compensation complex (DCC) of Drosophila identifies its X-chromosomal binding sites with exquisite selectivity. The principles that assure this vital targeting are known from the D. melanogaster model: DCC-intrinsic specificity of DNA binding, cooperativity with the CLAMP protein, and noncoding roX2 RNA transcribed from the X chromosome. We found that in D. virilis, a species separated from melanogaster by 40 million years of evolution, all principles are active but contribute differently to X specificity. In melanogaster, the DCC subunit MSL2 evolved intrinsic DNA-binding selectivity for rare PionX sites, which mark the X chromosome. In virilis, PionX motifs are abundant and not X-enriched. Accordingly, MSL2 lacks specific recognition. Here, roX2 RNA plays a more instructive role, counteracting a nonproductive interaction of CLAMP and modulating DCC binding selectivity. Remarkably, roX2 triggers a stable chromatin binding mode characteristic of DCC. Evidently, X-specific regulation is achieved by divergent evolution of protein, DNA, and RNA components.

PionX sites mark the X chromosome for dosage compensation.

The rules defining which small fraction of related DNA sequences can be selectively bound by a transcription factor are poorly understood. One of the most challenging tasks in DNA recognition is posed by dosage compensation systems that require the distinction between sex chromosomes and autosomes. In Drosophila melanogaster, the male-specific lethal dosage compensation complex (MSL-DCC) doubles the level of transcription from the single male X chromosome, but the nature of this selectivity is not known. Previous efforts to identify X-chromosome-specific target sequences were unsuccessful as the identified MSL recognition elements lacked discriminative power. Therefore, additional determinants such as co-factors, chromatin features, RNA and chromosome conformation have been proposed to refine targeting further. Here, using an in vitro genome-wide DNA binding assay, we show that recognition of the X chromosome is an intrinsic feature of the MSL-DCC. MSL2, the male-specific organizer of the complex, uses two distinct DNA interaction surfaces-the CXC and proline/basic-residue-rich domains-to identify complex DNA elements on the X chromosome. Specificity is provided by the CXC domain, which binds a novel motif defined by DNA sequence and shape. This motif characterizes a subclass of MSL2-binding sites, which we name PionX (pioneering sites on the X) as they appeared early during the recent evolution of an X chromosome in D. miranda and are the first chromosomal sites to be bound during de novo MSL-DCC assembly. Our data provide the first, to our knowledge, documented molecular mechanism through which the dosage compensation machinery distinguishes the X chromosome from an autosome. They highlight fundamental principles in the recognition of complex DNA elements by protein that will have a strong impact on many aspects of chromosome biology.

Two-step mechanism for selective incorporation of lncRNA into a chromatin modifier.

The MLE DExH helicase and the roX lncRNAs are essential components of the chromatin modifying Dosage Compensation Complex (DCC) in Drosophila. To explore the mechanism of ribonucleoprotein complex assembly, we developed vitRIP, an unbiased, transcriptome-wide in vitro assay that reveals RNA binding specificity. We found that MLE has intrinsic specificity for U-/A-rich sequences and tandem stem-loop structures and binds many RNAs beyond roX in vitro. The selectivity of the helicase for physiological substrates is further enhanced by the core DCC. Unwinding of roX2 by MLE induces a highly selective RNA binding surface in the unstructured C-terminus of the MSL2 subunit and triggers-specific association of MLE and roX2 with the core DCC. The exquisite selectivity of roX2 incorporation into the DCC thus originates from intimate cooperation between the helicase and the core DCC involving two distinct RNA selection principles and their mutual refinement.

Structural basis of X chromosome DNA recognition by the MSL2 CXC domain during Drosophila dosage compensation.

The male-specific lethal dosage compensation complex (MSL-DCC) selectively assembles on the X chromosome in Drosophila males and activates gene transcription by twofold through histone acetylation. An MSL recognition element (MRE) sequence motif nucleates the initial MSL association, but how it is recognized remains unknown. Here, we identified the CXC domain of MSL2 specifically recognizing the MRE motif and determined its crystal structure bound to specific and nonspecific DNAs. The CXC domain primarily contacts one strand of DNA duplex and employs a single arginine to directly read out dinucleotide sequences from the minor groove. The arginine is flexible when bound to nonspecific sequences. The core region of the MRE motif harbors two binding sites on opposite strands that can cooperatively recruit a CXC dimer. Specific DNA-binding mutants of MSL2 are impaired in MRE binding and X chromosome localization in vivo. Our results reveal multiple dynamic DNA-binding modes of the CXC domain that target the MSL-DCC to X chromosomes.

Looking through the FOG: microbiome characterization and lipolytic bacteria isolation from a fatberg site.

Sewer systems are complex physical, chemical and microbial ecosystems where fats, oils and grease (FOG) present a major problem for sewer management. Their accumulation can lead to blockages ('Fatbergs'), sewer overflows and disruption of downstream wastewater treatment. Further advancements of biological FOG treatments need to be tailored to degrade the FOG, and operate successfully within the sewer environment. In this study we developed a pipeline for isolation of lipolytic strains directly from two FOG blockage sites in the UK, and isolated a range of highly lipolytic bacteria. We selected the five most lipolytic strains using Rhodamine B agar plates and pNP-Fatty acid substrates, with two Serratia spp., two Klebsiella spp. and an environmental Acinetobacter strain that all have the capacity to grow on FOG-based carbon sources. Their genome sequences identified the genetic capacity for fatty acid harvesting (lipases), catabolism and utilization (Fad genes). Furthermore, we performed a preliminary molecular characterization of the microbial community at these sites, showing a diverse community of environmental bacteria at each site, but which did include evidence of sequences related to our isolates. This study provides proof of concept to isolation strategies targeting Fatberg sites to yield candidate strains with bioremediation potential for FOG in the wastewater network. Our work sets the foundation for development of novel bioadditions tailored to the environment with non-pathogenic Acinetobacter identified as a candidate for this purpose.

MSL2 combines sensor and effector functions in homeostatic control of the Drosophila dosage compensation machinery.

The process of dosage compensation (DC) in Drosophila counterbalances the monosomy of the X chromosome in male flies by increasing the transcription from this unique chromosome in the two-fold range. Upon exclusive expression of male-specific lethal 2 (MSL2) in males, the dosage compensation machinery assembles on active X-chromosomal genes. Overexpression of MSL proteins leads to aberrant binding of complex components to autosomes. Accordingly, MSL levels have to be carefully regulated. Here we describe a new mechanism through which MSL2 can fulfill its role as the central regulator of the faithful biogenesis and functionality of the DC machinery. MSL2 is an E3 ligase that ubiquitylates itself and the other associated components when their stoichiometry is unbalanced, uncovering proteasome-dependent degradation as an additional layer of homeostatic control of MSL levels. Furthermore, systematic mapping of modification sites by mass spectrometry and chromatin interaction studies on the target protein MSL1 suggest that the role of MSL2-mediated ubiquitylation goes beyond proteolysis.

Potential influence of sewer heat recovery on in-sewer processes.

Heat recovery from combined sewers has a significant potential for practical renewable energy provision as sources of heat demand and sewer pipes are spread across urban areas. Sewers are continuously recharged with relatively hot wastewater, as well as interacting with heat sources from surrounding air and soil. However, the potential effects of modifying sewage temperature on in-sewer processes have received little attention. The deposition of fats, oils and greases (FOGs) and hydrogen sulphide formation are biochemical processes and are thus influenced by temperature. This paper utilises a case study approach to simulate anticipated temperature reductions in a sewer network due to heat recovery. A laboratory investigation into the formation of FOG deposits at temperatures varying between 5 °C and 20 °C provided mixed results, with only a weak temperature influence, highlighting the need for more research to fully understand the influence of the wastewater composition as well as temperature on FOG deposit formation. A separate modelling investigation into the formation of hydrogen sulphide when inflow temperature is varied between 5 °C and 20 °C showed considerable reductions in hydrogen sulphide formation. Hence, heat extraction from sewers could be a promising method for managing some in-sewer processes, combined with traditional methods such as chemical dosing.

Role of the polycomb repressive complex 2 in acute promyelocytic leukemia.

Epigenetic changes are common alterations in cancer cells. Here, we have investigated the role of Polycomb group proteins in the establishment and maintenance of the aberrant silencing of tumor suppressor genes during transformation induced by the leukemia-associated PML-RARalpha fusion protein. We show that in leukemic cells knockdown of SUZ12, a key component of Polycomb repressive complex 2 (PRC2), reverts not only histone modification but also induces DNA demethylation of PML-RARalpha target genes. This results in promoter reactivation and granulocytic differentiation. Importantly, the epigenetic alterations caused by PML-RARalpha can be reverted by retinoic acid treatment of primary blasts from leukemic patients. Our results demonstrate that the direct targeting of Polycomb group proteins by an oncogene plays a key role during carcinogenesis.

Inhibitory mechanisms on dry anaerobic digestion: Ammonia, hydrogen and propionic acid relationship.

Inhibitory pathways in dry anaerobic digestion are still understudied and current knowledge on wet processes cannot be easily transferred. This study forced instability in pilot-scale digesters by operating at short retention times (40 and 33 days) in order to understand inhibition pathways over long term operation (145 days). The first sign of inhibition at elevated total ammonia concentrations (8 g/l) was a headspace hydrogen level over the thermodynamic limit for propionic degradation, causing propionic accumulation. The combined inhibitory effect of propionic and ammonia accumulation resulted in further increased hydrogen partial pressures and n-butyric accumulation. The relative abundance of Methanosarcina increased while that of Methanoculleus decreased as digestion deteriorated. It was hypothesized that high ammonia, total solids and organic loading rate inhibited syntrophic acetate oxidisers, increasing their doubling time and resulting in its wash out, which in turn inhibited hydrogenotrophic methanogenesis and shifted the predominant methanogenic pathway towards acetoclastic methanogenesis at free ammonia over 1.5 g/l. C/N increases to 25 and 29 reduced inhibitors accumulation but did not avoid inhibition or the washout of syntrophic acetate oxidising bacteria.

Feasibility and safety of adoptive immunotherapy with ex vivo-generated autologous, cytotoxic T lymphocytes in patients with solid tumor.

BACKGROUND AIMS: Adoptive T-cell therapy with tumor-specific T cells has emerged as a potentially useful approach for treating patients with advanced malignancies. We have demonstrated previously the feasibility of obtaining large numbers of autologous anti-tumor-specific cytotoxic T lymphocytes (CTL) generated by stimulation of patients' peripheral blood mononuclear cells with dendritic cells pulsed with apoptotic tumor cells. Methods. Six patients with progressing metastatic solid tumors (one renal cell carcinoma, two ovarian cancers, two extraosseous peripheral neuroectodermal tumors, one soft tissue sarcoma) not eligible for conventional therapies were treated with adoptive immunotherapy. Anti-tumor CTL, proven to be reactive in vitro against patient tumor cells, but not against normal cells, were infused following lymphodepleting chemotherapy administered to favor T-cell proliferation in vivo. RESULTS: Patients received a median of nine CTL infusions (range 2-19). The median number of CTL administered per infusion was 11 × 10(8) (range 1-55 × 10(8)). No patient experienced acute or late adverse events related to CTL infusion, even when large numbers of cells were given. Post-infusion laboratory investigations demonstrated an increase in the frequency of circulating anti-tumor T-cells and, in patients with a longer follow-up receiving two CTL infusions/year, a stabilization of these values. CONCLUSIONS: Our study demonstrates that autologous ex vivo-generated anti-tumor CTL can be administered safely in patients with advanced solid tumors and can improve the immunologic reactivity of recipients against tumor. These preliminary results provide a rationale for evaluating the clinical efficacy of this immunotherapeutic approach in phase I/II studies.

Characterisation and energy assessment of fats, oils and greases (FOG) waste at catchment level.

Several of the waste materials that have a negative impact on the sewer system are produced by fats, oils and greases (FOG) discharged from commercial and domestic kitchens. These materials accumulate at different points in the sewer catchment, from kitchens to pumping stations, sewers and sewage treatment works (STWs), and comprise oily wastewater, floating agglomerates and hard deposits. Despite their detrimental effects, these waste materials have a high calorific content and are an ideal feedstock for energy recovery processes. So far, the overall volume of each type of waste and their physical-chemical properties in relation to their collection point are unknown. However, from a management point of view, knowledge on each feedstock quality and volumes is necessary to develop an economic viable solution for their collection and for energy recovery purposes. In this study, FOG wastes collected from households, food service establishments (FSEs), sewage pumping stations, sewers and STWs, were compared to sewage sludge in terms of organic contents and energy potentials. As expected, FOG recovered at source (households and FSEs) were 'cleaner' and had a higher energy content. Once mixed with wastewater the materials changed in composition and lost some of their energy per unit mass. Our results showed that around 94,730 tonnes.year-1 of these materials could be recovered from the Thames Water Utilities' catchment, one of the most populated in the UK. These materials could produce up to 222 GWh.year-1 as biogas, close to double of what is produced with sewage sludge digestion and around 19% of the company energy needs. Finally, even with over six million households in the catchment, the results showed that most of the FOG waste was produced by FSEs (over 48,000 premises) with an estimated average of 79,810 tonnes.year-1 compared to 14,920 tonnes.year-1 from private households. This is an important outcome as recovery from FSEs will be cheaper and easier if the company decides to implement a collection system for energy recovery.

Achieving drinking water compliance levels for metaldehyde with an acclimated sand bioreactor.

Metaldehyde removal was delivered to below the 0.1 µg L-1 regulatory concentration in a laboratory scale continuous upflow fluidised sand bioreactor that had undergone acclimation through selective enrichment for metaldehyde degradation. This is the first reported case of successful continuous flow biological treatment of metaldehyde from real drinking water sources treating environmentally realistic metaldehyde concentrations. The impact of the acclimation process was impermanent, with the duration of effective treatment directly related to the elevated concentration of metaldehyde used during the enrichment process. The efficacy of the approach was demonstrated in continuous flow columns at both laboratory and pilot scale enabling degradation rates of between 0.1 and 0.2 mg L-1 h-1. Future work needs to focus on optimisation of the sand bioreactor and the acclimation process to ensure viability and feasibility of the approach at full scale.

Assessing the potential of enhanced primary clarification to manage fats, oils and grease (FOG) at wastewater treatment works.

Daily, sewage treatment works (STWs) receive large volumes of fats, oils and greases (FOG), by-products of food preparation. To increase FOG removal at STW, conventional primary sedimentation tanks (PSTs) can be enhanced using chemical coagulant or through dissolved air flotation (DAF) techniques. This work aimed to assess the potential benefits of enhanced primary treatment for FOG removal through an energy and costs analysis. To achieve this, a five-year sampling programme was conducted monthly at 15 STWs measuring FOG concentrations in crude and settled sewage (i.e. after primary treatment). In addition, two DAF pilot systems were trialled for four months and their performance, in terms of FOG removal, was assessed and compared to that of a control primary clarifier. Across the 15 STWs, influent FOG concentrations were found at 57 ±â€¯11 mg.L-1. Chemical coagulants dosed prior to PSTs increased FOG removal rates on average to 71% whilst traditional sedimentation only achieved 50% removal. Effluent FOG concentrations were found between 12-22 mg.L-1 and 19-36 mg.L-1 respectively. By contrast, DAF achieved FOG effluent concentrations on average at 10 ±â€¯4 mg.L-1 corresponding to 74% removal from a relatively low influent concentration of 40 ±â€¯30 mg.L-1. Thus, enhanced primary treatments have the potential to reduce organic load to secondary treatment and increase energy generation through anaerobic digestion. The overall net energy balance was estimated at 2269 MWh.year-1 for the DAF compared to 3445 MWh.year-1 for the chemically-enhanced PST making it a less financially attractive alternative. Yet, in the case where the works require upgrading to accommodate flow or load increases, DAF appeared as a sensible option over sedimentation offering significantly lower capital costs and footprint. In relation to FOG management, upgrading all STWs is not realistic and will require understanding where the benefits would be the highest.

Dry anaerobic digestion of organic waste: A review of operational parameters and their impact on process performance.

Dry digestion is a suitable technology for treating organic wastes with varying composition such as the organic fraction of municipal solids waste. Yet, there is a need for further research to overcome some of the disadvantages associated with the high total solids content of the process. Optimisation of inoculum to substrate ratio, feedstock composition and size, liquid recirculation, bed compaction and use of bulking agents are some of the parameters that need further investigation in batch dry anaerobic digestion, to limit localised inhibition effects and avoid process instability. In addition, further attention on the relation between feedstock composition, organic loading rate and mixing regimes is required for continuous dry anaerobic digestion systems. This paper highlights all the areas where knowledge is scarce and value can be added to increase dry anaerobic digestion performance and expansion.

Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors.

BACKGROUND: Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. DESIGN AND METHODS: We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. RESULTS: The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16(ink4a) protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-gamma secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E(2)-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. CONCLUSIONS: Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications.

Multiple mechanisms of telomere maintenance exist and differentially affect clinical outcome in diffuse malignant peritoneal mesothelioma.

PURPOSE: This study aims to investigate the prevalence of the two known telomere maintenance mechanisms, telomerase activity (TA) and alternative lengthening of telomeres (ALT), and to assess their prognostic relevance in diffuse malignant peritoneal mesothelioma (DMPM). EXPERIMENTAL DESIGN: In 44 DMPM specimens obtained from 38 patients, TA was determined using the telomeric repeat amplification protocol and ALT was detected by assaying ALT-associated promyelocytic leukemia nuclear bodies. The prognostic significance of telomere maintenance mechanisms was analyzed by Cox regression in the overall series and in a subset of 29 patients who underwent a uniform treatment regimen consisting of cytoreductive surgery and hyperthermic i.p. chemotherapy. RESULTS: Telomere maintenance mechanisms were detectable in 86.4% of DMPM: ALT or TA alone was found in 18.2% or 63.6% of lesions, respectively, whereas two cases (4.6%) were ALT+/TA+. TA and ALT proved to be inversely associated (P = 0.002). In the overall series, TA was prognostic for 4-year relapse (TA+ versus TA-, hazard ratio, 3.30; 95% confidence interval, 1.23-8.86; P = 0.018) and cancer-related death (TA+ versus TA-, hazard ratio, 3.56; 95% confidence interval, 1.03-12.51; P = 0.045), whereas ALT failed to significantly affect clinical outcome. These results held true also in the subset of patients submitted to uniform treatment with cytoreductive surgery and hyperthermic i.p. chemotherapy. CONCLUSIONS: Our results indicate that both known telomere maintenance mechanisms, TA and ALT, are present in DMPM and differentially affect patient prognosis.

Human bone marrow derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms.

Significant improvement in the understanding of mesenchymal stem cell (MSC) biology has opened the way to their clinical use. However, concerns regarding the possibility that MSCs undergo malignant transformation have been raised. We investigated the susceptibility to transformation of human bone marrow (BM)-derived MSCs at different in vitro culture time points. MSCs were isolated from BM of 10 healthy donors and propagated in vitro until reaching either senescence or passage (P) 25. MSCs in the senescence phase were closely monitored for 8 to 12 weeks before interrupting the cultures. The genetic characterization of MSCs was investigated through array-comparative genomic hybridization (array-CGH), conventional karyotyping, and subtelomeric fluorescent in situ hybridization analysis both before and after prolonged culture. MSCs were tested for the expression of telomerase activity, human telomerase reverse transcriptase (hTERT) transcripts, and alternative lengthening of telomere (ALT) mechanism at different passages. A huge variability in terms of proliferative capacity and MSCs life span was noted between donors. In eight of 10 donors, MSCs displayed a progressive decrease in proliferative capacity until reaching senescence. In the remaining two MSC samples, the cultures were interrupted at P25 to pursue data analysis. Array-CGH and cytogenetic analyses showed that MSCs expanded in vitro did not show chromosomal abnormalities. Telomerase activity and hTERT transcripts were not expressed in any of the examined cultures and telomeres shortened during the culture period. ALT was not evidenced in the MSCs tested. BM-derived MSCs can be safely expanded in vitro and are not susceptible to malignant transformation, thus rendering these cells suitable for cell therapy approaches.

From full-scale biofilters to bioreactors: Engineering biological metaldehyde removal.

Polar, low molecular weight pesticides such as metaldehyde are challenging and costly to remove from drinking water using conventional treatment methods. Although biological treatments can be effective at treating micropollutants, through biodegradation and sorption processes, only some operational biofilters have shown the ability to remove metaldehyde. As sorption plays a minor role for such polar organic micropollutants, biodegradation is therefore likely to be the main removal pathway. In this work, the biodegradation of metaldehyde was monitored, and assessed, in an operational slow sand filter. Long-term data showed that metaldehyde degradation improved when inlet concentrations increased. A comparison of inactive and active sand batch reactors showed that metaldehyde removal happened mainly through biodegradation and that the removal rates were greater after the biofilm was acclimated through exposure to high metaldehyde concentrations. This suggested that metaldehyde removal was reliant on enrichment and that the process could be engineered to decrease treatment times (from days to hours). Through-flow experiments using fluidised bed reactors, showed the same behaviour following metaldehyde acclimation. A 40% increase in metaldehyde removal was observed in acclimated compared with non-acclimated columns. This increase was sustained for >40 days, achieving an average of 80% removal and compliance (<0.1 µâ€¯L-1) for >20 days. An initial microbial analysis of the acclimated and non-acclimated biofilm from the same filter materials, showed that the microbial community in acclimated sand was significantly different. This work presents a novel conceptual template for a faster, chemical free, low cost, biological treatment of metaldehyde and other polar pollutants in drinking water. In addition, this is the first study to report kinetics of metaldehyde degradation in an active microbial biofilm at a WTW.

Risk assessments for quality-assured, source-segregated composts and anaerobic digestates for a circular bioeconomy in the UK.

A circular economy relies on demonstrating the quality and environmental safety of wastes that are recovered and reused as products. Policy-level risk assessments, using generalised exposure scenarios, and informed by stakeholder communities have been used to appraise the acceptability of necessary changes to legislation, allowing wastes to be valued, reused and marketed. Through an extensive risk assessment exercise, summarised in this paper, we explore the burden of proof required to offer safety assurance to consumer and brand-sensitive food sectors in light of attempts to declassify, as wastes, quality-assured, source-segregated compost and anaerobic digestate products in the United Kingdom. We report the residual microbiological and chemical risks estimated for both products in land application scenarios and discuss these in the context of an emerging UK bioeconomy worth £52bn per annum. Using plausible worst case assumptions, as demanded by the quality food sector, risk estimates and hazard quotients were estimated to be low or negligible. For example, the human health risk of E. coli 0157 illness from exposure to microbial residuals in quality-assured composts, through a ready-to-eat vegetable consumption exposure route, was estimated at ~10-8 per person per annum. For anaerobic digestion residues, 7 × 10-3cases of E. coli 0157 were estimated per annum, a potential contribution of 0.0007% of total UK cases. Hazard quotients for potential chemical contaminants in both products were insufficient in magnitude to merit detailed quantitative risk assessments. Stakeholder engagement and expert review was also a substantive feature of this study. We conclude that quality-assured, source-segregated products applied to land, under UK quality protocols and waste processing standards, pose negligible risks to human, animal, environmental and crop receptors, providing that risk management controls set within the standards and protocols are adhered to.

Chemical behavior and in vitro activity of mixed phosphine gold(I) compounds on melanoma cell lines.

(31)P nuclear magnetic resonance and electrospray ionization-mass spectrometry studies on the melanoma cytotoxic chlorotriphenylphosphine-1,3-bis(diphenylphosphino)propanegold(I), [Au(DPPP)(PPh 3)Cl], show partial decomposition that includes the novel dinuclear cation [Au 2(DPPP) 2Cl] (+); its structure was calculated by using the density functional theory (DFT). Unexpectedly, by using the diphosphine ligand 1,2-bis(diphenylphosphino)ethane (DIPHOS), [{AuCl(PPh 3)} 2(mu 2-DIPHOS)] was obtained. Its X-ray crystal structure shows a unique triangular coordination sphere in contrast to the T-shaped geometry of related Au(I)-DIPHOS compounds. Its cytotoxic activity in JR8, SK-Mel-5, and 2/60 melanoma cell lines is dose-dependent and lower than that of [Au(DPPP)(PPh 3)Cl] because of its nonchelating nature. An in vitro study of the effect of both Au compounds on the B16V melanoma cell line gives credence to this structure-activity relationship. IC 50 indicates that both Au species are 10 times more active in B16V than in JR8, SK-Mel-5, and 2/60. Oxidation of [Au(DPPP)(PPh 3)Cl] toward Au(III) compounds and phosphine-oxides is observed upon reaction with hypochlorite in water/dimethyl sulfoxide solution, mimicking endogenous hypochlorite. A related reaction involving the formation of [AuCl 4] (-) is thermodynamically feasible according to DFT calculations.