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Zanthoxylum species are credited with various uses in ethnomedicine due to their rich metabolite composition. In Kenya, these include management of cancer and microbial related ailments. However, there are limited reports showing how the bioactivity of Kenyan Zanthoxylum species is linked to their phytochemical profiles. This study therefore aimed at examining the chemical variation among five Zanthoxylum species found in Kenya (Z. chalybeum, Z. gilletii, Z. holtzianum, Z. paracanthum and Z. usambarense) using metabolomics approaches and the anti-oxidant and antimicrobial activities of these species. In a Folin–Ciocalteu test, the phenolic content of the stem bark extracts of these species were 73.083–145.272 mg TAE/g, while the alkaloids (in bromothymol blue chromogenic test) and flavonoids (in aluminium chloride test) were found to be 152.39–207.19 mg ME/g, and 109.416–186.413 mg CE/g, respectively. These extracts also exerted strong antioxidant activities in the 2,2-iphenyl-1-picrylhydrazyl (DPPH) and ferric ion reducing antioxidant power assays. In a broth dilution assay, the extract of the stem bark of Z. holtzianum ability showed the highest antimicrobial activity, followed by Z. chalybeum stem bark extract. The activities were positively correlated to both flavonoids and alkaloids concentrations, while the concentration of phenolics had weak negative correlation to antimicrobial activities. A chemometric analysis of the liquid-chromatography mass spectrometry profiles led to grouping of the species into three clusters. This study illustrates the variation in the bioactivity of Zanthoxylum species based on metabolite composition and justifies the wide usage of Zanthoxylum species in Kenyan traditional medicinal practices. Graphical abstract

Abnormally high-odor detection threshold (DT) values were detected for biosolids produced at one of the water resource recovery facilities (WRRFs) of Washington Suburban Sanitary Commission. As an inexpensive countermeasure, aeration of thickened sludge holding tanks (SHTs) was tested as a solution for mitigating the subsequent biosolids odor emission. Experimental results indicated that the extremely low-oxidation reduction potential (ORP) in the SHTs and the fermentation of high-rate-activated sludge were primarily contributors to the odor emission from the dewatered cake. Two rounds of bench-scale experiments on different days confirmed that aerating the sludge in holding tanks reduced peak emission concentrations of sulfurous odorous compounds such as hydrogen sulfide (H2 S), methanethiol (MT), and dimethyl sulfide (DMS) from 203, 110, and 20 mg m-3 g-1 dry to 119, 70, and 14 mg m-3 g-1 dry, respectively. Further preliminary full-scale validation study showed that even a slight ORP improvement from -180 mV to -162 mV reduced the peak H2 S concentration from 87 to 48 mg m-3 g-1 dry and decreased the biosolids DT value from 4266 to 1862. It was concluded that lifting ORP in SHTs through aeration can be used by utilities as a simple means for biosolids odor control. PRACTITIONER POINTS: Anaerobic storage of high-rate active sludge was the main reason for the excessive biosolids odor. Aeration of sludge holding tanks can effectively reduce biosolids odor. A slight oxidation reduction potential improvement substantially reduced biosolids odor.

With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-a-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles (1-2) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis. The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29, with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29, and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis. Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time.

Although aluminum- and iron-based chemicals have been broadly used as the two most common types of coagulants for wastewater treatment, their impacts on the performance of downstream sludge management can be quite different and have not been well understood. This work reviewed and analyzed their similarities and differences in the context of the anaerobic digestion performance, dewaterability of digested sludge, and odor emission from dewatered biosolids. In short, iron-based coagulants tend to show less negative impact than aluminum-based coagulants. This can be attributed to the reduction of ferric to ferrous ions in the course of anaerobic digestion, which leads to a suite of changes in protein bioavailability, alkalinity and hydrogen sulfide levels, and in turn the sludge dewaterability and odor potential. Whether these observations still hold true in the context of thermally hydrolyzed sludge management remains to be studied. PRACTITIONER POINTS: The impacts of aluminum-/iron-based coagulant addition on municipal sludge anaerobic digestibility, dewaterability, and odor emission are reviewed. Iron-based coagulants show less negative impact on the sludge digestibility than aluminum-based coagulants. Conclusions may aid practitioners in selecting coagulants in practice and better understanding the mechanisms behind the phenomena.

With increasing drug resistance in tuberculosis (TB) patient populations, there is an urgent need for new drugs. Ideally, new agents should work through novel targets so that they are unencumbered by preexisting clinical resistance to current treatments. Benzofuran 1 was identified as a potential lead for TB inhibiting a novel target, the thioesterase domain of Pks13. Although, having promising activity against Mycobacterium tuberculosis, its main liability was inhibition of the hERG cardiac ion channel. This article describes the optimization of the series toward a preclinical candidate. Despite improvements in the hERG liability in vitro, when new compounds were assessed in ex vivo cardiotoxicity models, they still induced cardiac irregularities. Further series development was stopped because of concerns around an insufficient safety window. However, the demonstration of in vivo activity for multiple series members further validates Pks13 as an attractive novel target for antitubercular drugs and supports development of alternative chemotypes.

The distribution of phytochemicals and their contribution to antioxidant potentials in whole and dehulled Bambara groundnut (BGN) seeds was evaluated. Whole BGN seeds were sorted using the testa and hilium colour and further grouped into whole and dehulled BGN seeds. Extractions of both whole and dehulled BGN seeds was done using methanol and the extracts assayed for total phenolics (TPC), flavanol, flavonol, anthocyanin content, oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP). Methanolic extract of whole BGN seed exhibited higher flavanol and flavonol content as well as significantly higher in-vitro antioxidant activities than dehulled BGN seeds. The TPC of whole BGN seed extract ranged from 3.6 to 11.0 GAE/g, while that of dehulled BGN ranged from 2.7 to 3.2 GAE/g. Identification of phenolics in whole and dehulled BGN seed extract using UPLC-qTOF-MS, revealed the presence of monoterpenoids (iridoids), phenolic acids, flavonoids and lignans. Bivariate correlations showed anthocyanin demonstrated weak positive correlation between flavanol, flavonol and ORAC for whole BGN seed extract; and negative correlation between flavanol, TPC, FRAP and ORAC for dehulled BGN. Aside the effect of dehulling, whole BGN seeds exhibited the presence of phytochemicals with beneficial properties for food and industrial application.

In order to address the confounder of TBSA on burn outcomes, we sought to analyze our experience with the use of autologous skin cell suspensions (ASCS) in a cohort of subjects with hand burns whose TBSA totaled 20% or less. We hypothesized that the use of ASCS in conjunction with 2:1 meshed autograft for the treatment of hand burn injuries would provide comparable outcomes to hand burns treated with sheet or minimally meshed autograft alone. A retrospective review was conducted for all deep partial and full-thickness hand burns treated with split-thickness autograft (STAG) at our urban verified burn center between April 2018 and September 2020. The exclusion criterion was a TBSA greater than 20%. The cohorts were those subjects treated with ASCS in combination with STAG (ASCS(+)) vs those treated with STAG alone (ASCS(−)). All ASCS(+) subjects were treated with 2:1 meshed STAG and ASCS overspray while all ASCS(−) subjects had 1:1, piecrust, or unmeshed sheet graft alone. Outcomes measured included demographics, time to wound closure, proportion returning to work (RTW), and length of time to RTW. Mann–Whitney U test was used for comparisons of continuous variables and Fisher’s exact test for categorical variables. Values are reported as medians and 25th and 75th interquartile ranges. Fifty-one subjects fit the study criteria (ASCS(+) n = 31, ASCS(−) n = 20). The ASCS(+) group was significantly older than the ASCS(−) cohort (44 [32–54] vs 32 years [27.5–37], P = .009) with larger %TBSA burns (15% [9.5–17] vs 2% [1–4], P < .0001) and larger size hand burns (190 [120–349.5] vs 126 cm2 [73.5–182], P = .015). Comparable results were seen between ASCS(+) and ASCS(−), respectively, for time to wound closure (9 [7–13] vs 11.5 days [6.75–14], P = .63), proportion RTW (61% vs 70%, P = .56), and days for RTW among those returning (35 [28.5–57] vs 33 [20.25–59], P = .52). The ASCS(+) group had two graft infections with no reoperations, while ASCS(−) had one infection with one reoperation. No subjects in either group had a dermal substitute placed. Despite being significantly older, having larger hand wounds, and larger overall wounds within the parameters of the study criteria, patients with 20% TBSA burns or smaller whose hand burns were treated with 2:1 mesh and ASCS overspray had comparable time to wound closure, proportion of RTW, and time to return to work as subjects treated with 1:1 or piecrust meshed STAG. Our group plans to follow this work with scar assessments for a more granular picture of pliability and reconstructive needs.

The urease enzyme of Cryptococcus neoformans is linked to different metabolic pathways within the yeast cell, several of which are involved in polyamine metabolism. Cryptococcal biogenic amine production is, however, largely unexplored and is yet to be investigated in relation to urease. The aim of this study was therefore to explore and compare polyamine metabolism in wild-type, urease-negative and urease-reconstituted strains of C. neoformans. Mass spectrometry analysis showed that agmatine and spermidine were the major extra- and intracellular polyamines of C. neoformans and significant differences were observed between 26 and 37 °C. In addition, compared to the wild-type, the relative percentages of extracellular putrescine and spermidine were found to be lower and agmatine higher in cultures of the urease-deficient mutant. The inverse was true for intracellular spermidine and agmatine. Cyclohexylamine was a more potent polyamine inhibitor compared to DL-α-difluoromethylornithine and inhibitory effects were more pronounced at 37 °C than at 26 °C. At both temperatures, the urease-deficient mutant was less susceptible to cyclohexylamine treatment compared to the wild-type. For both inhibitors, growth inhibition was alleviated with polyamine supplementation. This study has provided novel insight into the polyamine metabolism of C. neoformans, highlighting the involvement of urease in biogenic amine production.

Dodonaea viscosa Jacq (Sapindaceae) is a medicinal plant with a worldwide distribution. The species has undergone enormous taxonomic changes which caused confusion amongst plant users. In Kenya, for example, two varieties are known to exist based on morphology, i.e., D. viscosa var. viscosa along the coast, and D. viscosa var. angustifolia in the Kenyan inland. These two taxa are recognized as distinct species in some reports. This prompted us to apply metabolomics to understand the relationship among naturally occurring populations of D. viscosa in Kenya, and to identify compounds that can assist in taxonomic delineation of the different varieties of D. viscosa from different parts of Kenya. The phytochemical variability of Kenyan D. viscosa var. angustifolia populations collected from four different geographical regions (Nanyuki, Machakos, Nairobi, and Narok) and one coastal D. viscosa var. viscosa (the Gazi) were analyzed by LC-MS using a metabolomics-driven approach. Four known compounds, two diterpenoids (dodonic acid (1), hautriwaic acid lactone (3), and two flavonoids (5,7,4&prime, 5&prime, tetrahydroxy-3,6,2&prime, trimethoxyflavone (2) and catechin (4)) were isolated and purified from the Gazi coastal collection. The presence of these compounds and their relative abundance in other populations was determined by LC-MS analyses. Multivariate statistical analyses of LC-MS data was used for the visualization of the patterns of variation and identification of additional compounds. Eleven discriminant compounds responsible for separating chemometric clusters were tentatively identified. In an antimicrobial assay, hautriwaic acid lactone (3) and catechin (4) were the most active compounds followed by the extract from the coastal (Gazi) population. The clustering pattern of the five populations of D. viscosa suggested that the metabolite profiles were influenced by geo-environmental conditions and did not support the current classification of D. viscosa based on morphology. This study disputes the current classification of D. viscosa in Kenya and recommends revision using tools such as molecular phylogenetics.

Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington's disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

Partner and localizer of BRCA2 (PALB2), is a principal component of Homologous Recombination Repair (HRR) facilitating recruitment of HRR proteins such as BRCA1, BRCA2 and Rad51. Inherited inactiva...

The European Bathing Water Directive (BWD; 76/160/EEC 2006) requires the implementation of early warning systems for bathing waters which are subject to short-term pollution events. To this end, the EU SWIM project is developing coastal water quality prediction models and alert systems at nine beach sites in the Republic of Ireland and Northern Ireland, which represent a range of baseline water quality and site conditions.At each site, statistical / machine-learning predictive models are being developed based on their site-specific relationships between fecal indicator bacteria and multiple environmental variables. A unique aspect of the approach being developed is the use of a historical back-cast climate data (Met Éireann's MÉRA dataset) as the foundation of model development, and the use of a related climate forecast dataset (Met Éireann's Harmonie dataset) for forecasts. By integrating these datasets into a predictive system, environmental variables can be utilized at spatial and temporal resolutions exceeding what is typically available from alternative data sources (e.g. weather station gauges). This approach enables the production of a continuous stream of short-term water quality forecasts, which can then be validated against data collected by routine compliance sampling, as well as targeted supplementary water quality sampling.This presentation provides an overview of the end-to-end prediction system, a summary of the underlying models, and a discussion of the challenges and opportunities presented by this forecasting framework.

Consider α fetoprotein testing in later-onset ataxia as a screening marker as, if elevated, the differential diagnosis narrows considerably.

Wildly grown in most regions of the world, Carissa edulis is a highly underutilised fruit with significant antioxidant characteristics. The phyto and physicochemical properties of C. edulis berries at different stages of ripening are evaluated in this work. Total flavonoids (TF), total phenolic content (TPC) and antioxidant activity were determined spectrophotometrically, while concentration of polyphenols was determined using liquid chromatography coupled to diode array detection and electrospray ionization mass spectrometry. Results showed that antioxidant activity was lowest (18.36 &plusmn, 0.12 mg TE/g) in RS3 and decreased with TPC upon increased ripening. Conversely, TF increased with ripening progression with TF found to be highest in RS3 (5.92 &plusmn, 0.03 mg CE/g). Identified phenolic acids in C. edulis were quinic acid, protocatechuoyl-hexose, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and dicaffeoylquinic acid. Identified flavonoids included rutin, catechin, procyanidin dimer, procyanidin trimer, quercetin-3-O-glucosyl-xyloside, quercetin-3-O-robinobioside, quercetin-3-O-glucoside and quercetin-3-OH-3-methylglutaryl-glucoside. Physicochemical properties of C. edulis varied among samples with sugar/acid ratio of C. edulis ranging from 25.70 for RS1 to 50.36 for RS3. Ripening stage of C. edulis undoubtedly affects the phyto and physicochemical properties of C. edulis.

Vannbårne smitteutbrudd forekommer i avanserte rense- og distribusjonssystemer og det siste store smitteutbruddet i Norge fant sted våren 2019 i Askøy kommune. Smitteutbrudd er uønsket da de kan gi alvorlige helsekonsekvenser og i værste fall føre til død. I dag kontrolleres kvaliteten til norskdrikkevannmedindikatororgansimer,mensporadiskesmittutbrudder et tegn på at kvalitetskontrollen kan bli bedre. Verdens helseorganisasjon (WHO) anbefaler å utvide kvalitetskontrollen med forebyggende risikovurderinger og nevner kvantitativ mikrobiell risikovurdering (QMRA) som et godt alternativ. Denne oppgaven fortsetter på arbeidet til Svanemyr (2019). Et program ble skrevetslikatdetvarmuligåutførerisikovurderingerfordistribusjonsnettet tilSpydebergkommune.ProgrammeterbasertpåepanetklasseniEPANETMATLAB Toolkit og bruker EPANET-MSX til å simulere spredningen av patogener i ledningsnettverket. Programmet fungerer uten større problemer, men nettmodellen og risikovurdering er avhengig av mer arbeid før det er mulig å si at resultatene er representative for en forurensningshendelse i Spydeberg kommune. Risikovurderingen returnerer punktestimater som bør brukes med forsiktighet. Folkehelseinstituttet (FHI) har en pågående drikkevannsstudie, der et av målene er å undersøke sammenhengen mellom trykkløsepisoder i ledningsnettverket og gastroenteritt i befolkningen. Forhåpentligvis kan informasjon fra studiet brukes til å sammenligne epidemiologiske data med teoretiske resultater fra risikovurderinger utført på ledningsnettverket til Spydeberg. Water-borneinfectionoutbreaksoccurinadvancedcleaninganddistribution systems The last major outbreak in Norway took place in Askøy municipality this spring (2019). Infection outbreaks are undesirable as they can have a serious impact on people’s health. Today, the quality of Norwegian drinking water is controlled with indicator organisms. Unfortunately, occasional outbreaks indicate that quality control could be improved. The World Health Organization (WHO) recommends expanding quality control with preventive risk assessments and mentions quantitative microbial risk assessment (QMRA) as a good alternative. This thesis is a continuation of Svanemyr (2019) work. A program was written to carry out risk assessments for the distribution system in Spydeberg. TheprogramisbasedontheepanetclassintheEPANET-MATLABToolkit and uses EPANET-MSX to simulate the spread of pathogens. The program works without major problems, but the distribution model and risk assessment need more work before the results are representative for Spydeberg municipality. The risk assessment returns point estimates that should be used with caution. The Norwegian Institute of Public Health (NIPH) has an ongoing drinking water study where one of the goals is to determine the relationship between lossofpressureandgastrointestinaldiseases.Hopefullyinformationfromthe study can be used to compare epidemiological data with theoretical results from the QMRA performed on Spydeberg’s distribution system. M-VM

The production of hydraulic fracturing fluids (HFFs) in natural gas extraction and their subsequent management results in waste streams highly variable in total dissolved solids (TDS). Because TDS measurement is time-consuming, it is often estimated from electrical conductivity (EC) assuming dissolved solids are predominantly ionic species of low enough concentration to yield a linear TDS-EC relationship: TDS (mg/L) = ke × EC (μS/cm) where ke is a constant of proportionality. HHFs can have TDS levels from 20,000 to over 300,000 mg/L wherein ion-pair formation and non-ionized solutes invalidate a simple TDS-EC relationship. Therefore, the composition and TDS-EC relationship of several fluids from Marcellus gas wells in Pennsylvania were assessed. Below EC of 75,000 μS/cm, TDS (mg/L) can be estimated with little error assuming ke = 0.7. For more concentrated HFFs, a curvilinear relationship (R2 = 0.99) is needed: TDS = 27,078e1.05 × 10−5*EC. For hypersaline HFFs, the use of an EC/TDS meter underestimates TDS by as much as 50%. A single linear relationship is unreliable as a predictor of brine strength and, in turn, potential water quality and soil impacts from accidental releases or the suitability of HFFs for industrial wastewater treatment.

TP53 and ataxia telangiectasia mutated (ATM) defects are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukemia (CLL). Currently, therapies capable of providing durable remissions in relapsed/refractory TP53- or ATM-defective CLL are lacking. Ataxia telangiectasia and Rad3-related (ATR) mediates response to replication stress, the absence of which leads to collapse of stalled replication forks into chromatid fragments that require resolution through the ATM/p53 pathway. Here, using AZD6738, a novel ATR kinase inhibitor, we investigated ATR inhibition as a synthetically lethal strategy to target CLL cells with TP53 or ATM defects. Irrespective of TP53 or ATM status, induction of CLL cell proliferation upregulated ATR protein, which then became activated in response to replication stress. In TP53- or ATM-defective CLL cells, inhibition of ATR signaling by AZD6738 led to an accumulation of unrepaired DNA damage, which was carried through into mitosis because of defective cell cycle checkpoints, resulting in cell death by mitotic catastrophe. Consequently, AZD6738 was selectively cytotoxic to both TP53- and ATM-defective CLL cell lines and primary cells. This was confirmed in vivo using primary xenograft models of TP53- or ATM-defective CLL, where treatment with AZD6738 resulted in decreased tumor load and reduction in the proportion of CLL cells with such defects. Moreover, AZD6738 sensitized TP53- or ATM-defective primary CLL cells to chemotherapy and ibrutinib. Our findings suggest that ATR is a promising therapeutic target for TP53- or ATM-defective CLL that warrants clinical investigation.

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. However, they are vulnerable parts of the landscapes that are often poorly understood and understudied. Land use and management practices, as well as climate change are thought to be a contribution to the global amphibian decline. However, more research is needed to understand the extent of these impacts. Here, we present methodology for characterizing a vernal pond's morphology and detail a monitoring station that can be used to collect water quantity and quality data over the duration of a vernal pond's hydroperiod. We provide methodology for how to conduct field surveys to characterize the morphology and develop stage-storage curves for a vernal pond. Additionally, we provide methodology for monitoring the water level, temperature, pH, oxidation-reduction potential, dissolved oxygen, and electrical conductivity of water in a vernal pond, as well as monitoring rainfall data. This information can be used to better quantify the ecosystem services that vernal ponds provide and the impacts of anthropogenic activities on their ability to provide these services.

Tea samples from 17 populations of "wild tea" ecotypes Aspalathus linearis (rooibos tea) and 2 populations of Aspalathus pendula were analyzed. Recent advances in column technology together with high-resolution mass spectrometry were applied to improve resolution, facilitating the identification of several new compounds as well as grouping of the wild tea ecotypes according to their chemical composition. The collisional cross-section data obtained from ion mobility-mass spectrometry is reported for the flavonoids in rooibos for the first time. Enzyme pathways for the synthesis of the unique flavonoids found in rooibos tea are also proposed. A. linearis and A. pendula produce similar combinations of main phenolic compounds, with no diagnostically different discontinuities between populations or species. Northern resprouters (Gifberg and Nieuwoudtville) contain higher phenylpropenoic acid glucoside levels while teas from Wupperthal and surrounding areas were found to contain unique dihydrochalcones (phloridzin and a sieboldin analog), which are reported here for the first time.

Stabilization and dewatering methods for wastewater solids determine the concentration and nature of phosphorus (P) in biosolids and in-plant sidestreams recycled to the liquid treatment facility. Because water treatment residuals (WTR) exhibit strong immobilization of soluble P, this study evaluated the impact of co-dewatering WTR and biosolids on the P partitioning during dewatering and the environmental lability of biosolids-P measured by water-extractable P (WEP). Overall, P progressively partitioned into the water-insoluble particulate-bound form in dewatered cake with increasing blending ratio (BR) – defined as the dry mass ratio of WTR to biosolids. The reject water total P (TP) content from dewatering biosolids alone (250 mg L−1) was reduced to 60 mg L−1 for a BR = 1.5. Polymer addition resulted in statistically (α = 0.05) lower reject liquid TP, suggesting the cationic polyelectrolyte contributed to P binding. The WEP of the dewatered cake (∼20% solids) dropped from 2.36 g kg−1 (biosolids only) to ∼0.14 g kg−1 for BR = 1.5, meaning the P in land-applied co-processed cake is less susceptible to solubilization by surface runoff compared to unamended biosolids. Co-dewatering can reduce P in return flows and fix P in the dewatered solids in a form less prone to off-site migration following land application.

ATM is a principal DNA damage response protein that synchronises a complex network of cellular responses to double stranded DNA breaks. ATM gene is recurrently mutated in a wide range of lymphoid malignancies, including B-cell chronic lymphocytic leukemia (CLL), T-prolymphocytic leukaemia (T-PLL), mantle cell lymphoma (MCL) and diffuse B cell lymphoma (DLBCL). ATM pathway is utilized by many DNA damaging agents and consequently inactivation of this pathway can lead to chemoresistance. Furthermore, in the absence of ATM tumour cells exhibit genomic instability that can lead to clonal selection and evolution even under current targeted treatments. Consequently there is clear need to understand dependency pathways in ATM-deficient tumours and apply tailored targeted therapies that will specifically eliminate those tumour cells. We have previously presented a novel murine model of ATM-deficiency that spontaneously generate lymphoid tumours, mostly DLBCL. These tumours have been successfully propagated both in recipient mice and in vitro, where several cell lines have been generated. Genome editing methods, such as CRISPR/CAS-9, permit the targeted disruption of specific genes. Protocols for genome wide screens have been developed based on this technology which can be used to identify genes that are essential for cellular survival. As such, these screens can be used to identify dependency pathways for tumours with specific genetic lesions. Using lentiviral transduction we established two cell lines that stably expressed CAS-9. We then performed a genome wide CRISPR screen using the GeCKO library to identify novel therapeutic targets in these Atm-deficient tumours. This library consists of 130,209 unique single guide RNA (sgRNAs), targetting 20,611 genes including 1176 miRNAs. A comparative analysis was performed of sgRNA drop-out following 15 cellular doublings. This revealed a number of pathways including those already known to be synthetically lethal with ATM deficiency, such as ATR and PARP. Pathway analysis of the top genes from this drop-out analysis identified oxidative phosphorylation, the spliceosome, ribosome biogenesis, N-glycan biosynthesis, pyrimidine metabolism and purine metabolism as the most significantly affected pathways. Furthermore, the drop-out screen revealed a number of miRNAs, including MiR-3470a, Mir-3971, MiR-669f and MiR-719. These data provide a unique molecular assessment of the dependency of ATM-deficient lymphomas and provide a number of novel putative therapeutic targets for treating such tumours. Disclosures No relevant conflicts of interest to declare.

Background : Acute leukemia is the most common cancer in children under 15 years of age; 80% are acute lymphoblastic leukemia (ALL) and 17% are acute myeloid leukemia (AML). Childhood leukemia shows further diversity based on cytogenetic and molecular characteristics, which may relate to distinct etiologies. Case–control studies conducted worldwide, particularly of ALL, have collected a wealth of data on potential risk factors and in some studies, biospecimens. There is growing evidence for the role of infectious/immunologic factors, fetal growth, and several environmental factors in the etiology of childhood ALL. The risk of childhood leukemia, like other complex diseases, is likely to be influenced both by independent and interactive effects of genes and environmental exposures. While some studies have analyzed the role of genetic variants, few have been sufficiently powered to investigate gene–environment interactions. Objectives : The Childhood Leukemia International Consortium (CLIC) was established in 2007 to promote investigations of rarer exposures, gene–environment interactions and subtype-specific associations through the pooling of data from independent studies. Methods : By September 2012, CLIC included 22 studies (recruitment period: 1962–present) from 12 countries, totaling approximately 31000 cases and 50000 controls. Of these, 19 case–control studies have collected detailed epidemiologic data, and DNA samples have been collected from children and child–parent trios in 15 and 13 of these studies, respectively. Two registry-based studies and one study comprising hospital records routinely obtained at birth and/or diagnosis have limited interview data or biospecimens. Conclusions : CLIC provides a unique opportunity to fill gaps in knowledge about the role of environmental and genetic risk factors, critical windows of exposure, the effects of gene–environment interactions and associations among specific leukemia subtypes in different ethnic groups.

We recently reported that two of six HLA-DP supertypes (DP1-4, 6, 8) were associated with susceptibility (DP2) and resistance (DP1) to childhood acute lymphoblastic leukaemia (ALL). To determine whether DP supertypes are associated with childhood ALL prognosis, we compared treatment outcomes in a cohort (n = 798) of DPB1-typed ALL cases in the UK Medical Research Council UKALL XI trial. No differences in clinical characteristics and outcome between DPB1-typed and untyped (n = 1292) cases suggest no selection bias. Event-free survival (EFS) rates in patients with DP1 and DP3 supertypes were significantly worse than in patients with DP2, DP4, DP6 and DP8 [10-year EFS: 55%; 95% confidence interval (CI) = 49-61%; compared with 64% (61-68%), P = 0.006]. Ten-year EFS in DP1/DP3 heterozygous patients [30% (2-58%)] was significantly worse than in patients with DP1, DP3 or neither allele [56% (50-62%); P = 0.02]. Lack of evidence that DP1 or DP3 are associated with known prognostic factors leads us to suggest that these two supertypes exert an independent effect on prognosis. This may involve abrogation of DP1/3-restricted T-cell control of residual disease due to selective effects of chemotherapy. Further studies of HLA supertypes in relation to outcome in recent childhood ALL trials may resolve this question.

Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the ‘diagnostic odyssey’ for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1–3, 6, 7 and Friedrich’s ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3–35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US$620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.

The human leukocyte antigen (HLA) genes are candidate genetic susceptibility loci for childhood acute lymphoblastic leukemia (ALL). We examined the effect of HLA-DP genetic variation on risk and evaluated its potential interaction with 4 proxies for early immune modulation, including measures of infectious exposures in infancy (presence of older siblings, daycare attendance, ear infections) and breastfeeding. A total of 585 ALL cases and 848 controls were genotyped at the HLA-DPA1 and DPB1 loci. Because of potential heterogeneity in effect by race/ethnicity, we included only non-Hispanic white (47%) and Hispanic (53%) children and considered these 2 groups separately in the analysis. Logistic regression analyses showed an increased risk of ALL associated with HLA-DPB1*01:01 (odds ratio [OR] = 1.43, 95% CI, 1.01-2.04) with no heterogeneity by Hispanic ethnicity (P = .969). Analyses of DPB1 supertypes showed a marked childhood ALL association with DP1, particularly for high-hyperdiploid ALL (OR = 1.83; 95% CI, 1.20-2.78). Evidence of interaction was found between DP1 and older sibling (P = .036), and between DP1 and breastfeeding (P = .094), with both showing statistically significant DP1 associations within the lower exposure categories only. These findings support an immune mechanism in the etiology of childhood ALL involving the HLA-DPB1 gene in the context of an insufficiently modulated immune system.

Ataxia-telangiectasia (A-T) is classically characterized by progressive neurodegeneration, oculocutaneous telangiectasia, immunodeficiency and elevated α-fetoprotein levels. Some patients, classified as variant A-T, exhibit a milder clinical course. In the latter patients extrapyramidal symptoms, instead of cerebellar ataxia, tend to be the dominating feature and other classical disease hallmarks, like telangiectasia, appear later or even may be absent. Some patients with variant disease have clinically pronounced anterior horn cell degeneration. Neuropathological studies of genetically proven A-T patients are lacking. The aims of our study were to describe the neuropathology of three A-T patients; in two of them the diagnosis was genetically confirmed. The neuropathological findings were compared with those of all known published autopsy findings in A-T patients up to now. Two classical A-T patients aged 19 and 22 and a 33-year-old patient with variant disease were autopsied. In line with previous reports, our patients had severe cerebellar atrophy, less pronounced degeneration of the dentate nucleus and inferior olive, degeneration of the posterior columns and neurogenic muscular atrophy. In addition, all three had anterior horn cell degeneration, which was most prominent at the lumbar level. Compared to the literature, the degenerative changes in the brain stem of the variant A-T patient were somewhat less than anticipated for his age. Degenerative changes in the cerebellum and spinal cord were comparable with those in the literature. Progeric changes were lacking. In conclusion, compared to classical A-T, the variant A-T patient showed essentially the same, only slightly milder neuropathological abnormalities, except for anterior horn degeneration.

Childhood B-cell precursor acute lymphoblastic leukemia (BCP ALL) is usually initiated in utero and is thought to progress to overt leukemia under the influence of delayed exposure to a common infection. Based on the hypothesis that polymorphic HLA-DP variants can restrict T-cell responses to infection, we previously compared DP supertype frequencies in BCP ALL patients with that of unrelated newborn controls. We reported that the DP2 supertype was associated with susceptibility, whereas DP1 was associated with protection. However, the association of genetic variants in children with early-onset diseases such as ALL may be a proxy for parental effects. Here we examine whether maternal DP1 and DP2 are associated with BCP ALL by fitting log-linear models in a combined series of family triads (both parents and case child) and dyads (1 parent and case child; n = 571) in comparison with similar models in non-BCP leukemia (n = 198). We report no evidence of maternal DP1 or DP2 associations with BCP ALL, but we did identify suggestive evidence of maternal undertransmission of the infrequent supertypes DP11 and DP15. Although these results require confirmation, they suggest that DP11 and DP15 may be protective or that there is transmission ratio distortion of these supertypes in BCP ALL.

The therapeutic exploitation of molecular defects within the DNA damage response (DDR) in tumour cells has become an important treatment paradigm. 'Synthetic lethality' relies on pharmacological inhibition of pathways upon which DDR-deficient tumour cells have become dependent for their survival. This induces an intolerable level of unrepaired DNA damage in the tumour cells resulting in cell death, whilst sparing DDR-proficient normal cells Deletion of 13q14 is a frequent, early event in the pathogenesis of CLL. Alongside well-described tumour suppressor genes this genomic region also encompasses the DDR gene, RNASEH2B, which encodes a subunit of the heterotrimeric enzymatic complex, RNaseH2. This complex is a principal component of ribonucleotide excision repair (RER), a DDR pathway that removes ribonucleotides incorporated into DNA by error prone DNA repair polymerases. If unremoved, these DNA-incorporated ribonucleotides lead to DNA damage, chromosome instability and mutagenesis (Reijns et al, Cell. 2012;149:1008). We recently reported a synthetically lethal interaction between the functional loss of RNaseH2 enzymatic complex and PARP inhibition (Zimmerman et al, Nature 2018, 559:285). We observed that inactivation of any of the three RNAseH2 subunits (A,B,C) leads to loss of enzymatic activity of this complex and also that primary CLL tumours with 13q14 deletion involving the RNASEH2B locus are sensitive to PARP inhibitors (PARPi) in vitro. In light of these preliminary observations, we addressed the following questions: a) Do monoalleic and biallelic RNASEH2B deletions have equal consequences for RNAseH2 enzymatic activity and sensitivity to PARP inhibition in CLL? d) Can loss of RNAseH2 activity be caused by an alternative mechanism, such as mutations in RNASEH2B? c) Can the PARPi sensitivity of RNaseH2-deficient CLLs be demonstrated in vivo, in patient-derived xenografts? d) Is PARP inhibition an option for RNAseH2 deficient tumours with limited response to other treatments? Analysis of 100 primary CLL tumours through a combination of multiplex ligation-dependent probe amplification (MLPA), CGH microarrays and Sanger sequencing identified 29 tumours with monoallelic and 14 with biallelic RNASEH2B deletions. None of the analysed tumours had mutations in RNASEH2B. Increased levels of genomic ribonucleotides were confirmed in all RNASEH2B deleted tumours by two complementary methods: alkaline gel electrophoresis and DNA nick translation. We found that the RNaseH2 enzymatic defect and sensitivity to PARP inhibition were evident in all RNASEH2B deleted tumours, but were more profound in those harbouring biallelic deletion compared to tumours that have lost only one RNASEH2B allele. Furthermore, sensitivity to PARP inhibitors was dependent on PARP-trapping capacity and therefore cytotoxicity was most prominent in response to PARP-inhibitors with a potent PARP trapping capacity such as talazoparib. In vivo experiments revealed similar trends, with CLL xenografts derived from tumours with biallelic RNASEH2B deletion being differentially sensitive to Talazoparib. Notably, the PARP inhibition sensitivity of RNAseH2-deficient primary CLLs was independent of patients' response to different treatments. In summary, we conclude that the RNASEH2B loss associated with 13q14 deletion represents a frequent cause of RNaseH2 enzymatic defect that renders primary CLL tumours sensitive to PARP-trapping inhibitors. Our findings expand the range of molecular defects in CLL that are amenable to treatment with clinically applicable PARP inhibitors and may have implications for the management of patients with limited response to other treatments. Disclosures No relevant conflicts of interest to declare.

Recent studies have reported that regions of homozygosity (ROH) in the genome are detectable in outbred populations and can be associated with an increased risk of malignancy. To examine whether homozygosity is associated with an increased risk of developing childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), we analyzed 824 ALL cases and 2398 controls genotyped for 292 200 tagging SNPs. Across the genome, cumulative distribution of ROH was not significantly different between cases and controls. Four common ROH at 10p11.2-10q11.21, 1p31.1, 19p13.2-3, and 20q11.1-23 were, however, associated with ALL risk at P less than .01 (including 1 ROH to which the erythropoietin receptor [EPOR] gene maps, P = .005) but were nonsignificant after adjusting for multiple testing. Our findings make it unlikely that levels of measured homozygosity, caused by autozygosity, uniparental isodisomy, or hemizygosity, play a major role in defining BCP-ALL risk in predominantly outbred populations.

Studies of childhood leukemia and the potential etiologic role of genetic variation in folate metabolism have produced conflicting findings and have often been based on small numbers. We investigated the association between polymorphisms in key folate metabolism enzymes (MTHFR 677 C>T, MTHFR 1298 A>C, SHMT1 1420 C>T, MTR 2756 A>G, TS 1494del6, and TS 28bp repeat) in 939 cases of childhood acute lymphoblastic leukemia (ALL) and 89 cases of acute myeloid leukemia (AML) recruited into the United Kingdom Childhood Cancer Study. We also examined the maternal genotypes of 752 of these cases. Data from 824 noncancer controls recruited were used for comparison. No evidence of an association with MTHFR 677 was observed for ALL or AML, either in children or their mothers. However, in children an increased risk of ALL (odds ratio [OR] = 1.88; 95% confidence interval [CI], 1.16-3.07; P = .010) and AML (OR = 2.74; 95% CI, 1.07-7.01; P = .036) was observed with the MTR 2756 GG genotype; the association was most pronounced for cases with the MLL translocation (OR = 4.90; 95% CI, 1.30-18.45; P = .019). These data suggest that genetic variation in methionine synthase could mediate risk of childhood leukemia, either via effects on DNA methylation or via effects on fetal growth and development.

Identification of de novo minisatellite mutations in the offspring of parents exposed to mutagenic agents offers a potentially sensitive measure of germ line genetic events induced by ionizing radiation and genotoxic chemicals. Germ line minisatellite mutations (GMM) are usually detected by hybridizing Southern blots of unamplified size-fractionated genomic DNA with minisatellite probes. However, this consumes a relatively large amount of DNA, requires several steps and may lack sensitivity. We have developed a polymerase chain reaction (PCR)-based GMM assay, which we applied to the hypermutable minisatellite, CEB1. Here, we compare the sensitivity and specificity of this assay with the conventional Southern hybridization method using DNA from 10 spouse pairs, one parent of each pair being a survivor of cancer in childhood, and their 20 offspring. We report that both methods have similar specificity but that the PCR method uses 250 times less DNA, has fewer steps and is better at detecting GMM with single repeats provided that specific guidelines for allele sizing are followed. The PCR GMM method is easier to apply to families where the amount of offspring DNA sample is limited.

To identify risk variants for childhood acute lymphoblastic leukemia (ALL), we conducted a genome-wide association study of two case-control series, analyzing the genotypes with respect to 291,423 tagging SNPs in a total of 907 ALL cases and 2,398 controls. We identified risk loci for ALL at 7p12.2 (IKZF1, rs4132601, odds ratio (OR) = 1.69, P = 1.20 x 10(-19)), 10q21.2 (ARID5B, rs7089424, OR = 1.65, P = 6.69 x 10(-19)) and 14q11.2 (CEBPE, rs2239633, OR = 1.34, P = 2.88 x 10(-7)). The 10q21.2 (ARID5B) risk association appears to be selective for the subset of B-cell precursor ALL with hyperdiploidy. These data show that common low-penetrance susceptibility alleles contribute to the risk of developing childhood ALL and provide new insight into disease causation of this specific hematological cancer. Notably, all three risk variants map to genes involved in transcriptional regulation and differentiation of B-cell progenitors.

The extended human major histocompatibility complex (MHC) is a gene-rich region of about 7.6 Mb on chromosome 6, and includes a high proportion of genes involved in the immune response. Among these are the two Human Leukocyte Antigen (HLA) gene clusters, class I and class II, which encode highly polymorphic classical HLA-A, B, C and HLA-DR, DQ and DP genes, respectively. The protein products of the classical HLA genes are heterodimeric cell surface molecules that bind short peptides derived from non-self and self proteins, including infections and auto-antigens. The presentation of these HLA-anchored peptides to T lymphocytes triggers a cascade of responses in immune-associated genes that leads to adaptive immunity. Associations between HLA class II alleles and childhood leukemia have been reported in a number of studies. This could be due to the role of HLA allele-restricted peptide binding and T cell activation, or linkage disequilibrium to an MHC-linked “leukemia gene” in the pathogenesis of childhood leukemia. Efforts are currently in progress to resolve these questions, using large leukemia case-control sample series such as the UK Childhood Cancer Study (UKCCS) and the Northern California Childhood Leukemia Study (NCCLS). Here we review the background to these studies, and present a novel hypothesis based on the paradigm of HLA-associated auto-immune disease that might explain an infection-based etiology of childhood leukemia.

Most childhood B cell precursor (BCP) acute lymphoblastic leukaemia (ALL) cases carry the reciprocal translocation t(12;21)(p13;q22) ( approximately 25%), or a high hyperdiploid (HeH) karyotype (30%). The t(12;21) translocation leads to the expression of a novel fusion gene, TEL-AML1 (ETV6-RUNX1), and HeH often involves tri- and tetrasomy for chromosome 21. The presence of TEL-AML1+ and HeH cells in utero prior to the development of leukaemia suggests that these lesions play a critical role in ALL initiation. Based on our previous analysis of HLA-DP in childhood ALL, and evidence from in vitro studies that TEL-AML1 can activate HLA-DP-restricted T cell responses, we hypothesised that the development of TEL-AML1+ ALL might be influenced by the child's DPB1 genotype. To test this, we analysed the frequency of six HLA-DPB1 supertypes in a population-based series of childhood leukaemias (n = 776) classified by their karyotype (TEL-AML1+, HeH and others), in comparison with newborn controls (n = 864). One DPB1 supertype (GKD) conferred significant protection against TEL-AML1+ ALL (odds ratio (OR), 95% confidence interval (95% CI): 0.42, 0.22-0.81; p < 0.005) and HeH ALL (OR; 95% CI: 0.44, 0.30-0.65; p < 0.0001). These negative associations were almost entirely due to a single allele, DPB1*0101. Our results suggest that DPB1*0101 may afford protection from the development of TEL-AML1+ and HeH BCP ALL, possibly as the result of a DP-restricted immune response to BCP ALL-associated antigen(s), the identification of which could have important implications for the design of prophylactic vaccines.