Optimization of TAM16, a Benzofuran That Inhibits the Thioesterase Activity of Pks13; Evaluation toward a Preclinical Candidate for a Novel Antituberculosis Clinical Target.

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.

Biallelic Mutation of SETX and Additional Likely "In Cis" SETX Sequence Change in Ataxia with Oculomotor Apraxia Type 2.

Ataxia with oculomotor apraxia type 2 (AOA2) is a slowly progressive, autosomal recessive disease characterized by the triad of ataxia, oculomotor apraxia, and sensorimotor neuropathy. The genetic basis of AOA2 is biallelic mutation of the SETX gene, resulting in reduced or absent senataxin, a DNA/RNA repair protein essential for genomic stability. In this case report, we described a case of AOA2 with two clear pathogenic SETX mutations, one of which is novel. We then discussed two further likely "in cis" SETX sequence changes (previously reported in the literature as pathogenic), and presented the case that they are likely benign polymorphisms.

A link between urease and polyamine metabolism in Cryptococcus neoformans.

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.

Phytochemical composition and antioxidant properties of methanolic extracts of whole and dehulled Bambara groundnut (Vigna subterranea) seeds.

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.

Aeration in sludge holding tanks as an economical means for biosolids odor control-A case study.

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.

Spirocycle MmpL3 Inhibitors with Improved hERG and Cytotoxicity Profiles as Inhibitors of Mycobacterium tuberculosis Growth.

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-à-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.

Impacts of aluminum- and iron-based coagulants on municipal sludge anaerobic digestibility, dewaterability, and odor emission.

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.

LC-MS-Based Metabolomics for the Chemosystematics of Kenyan Dodonaea viscosa Jacq (Sapindaceae) Populations.

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',5'-tetrahydroxy-3,6,2'-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.

Imaging Mutant Huntingtin Aggregates: Development of a Potential PET Ligand.

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.

Pharmacokinetic Interaction of Green Rooibos Extract With Atorvastatin and Metformin in Rats.

An aspalathin-rich green rooibos extract (Afriplex GRT™) has demonstrated anti-diabetic and hypolipidemic properties, while also moderately inhibiting CYP3A4 activity, suggesting a potential for herb-drug interaction. The present study, therefore, evaluated the effects of orally administered GRT on the pharmacokinetics of atorvastatin and metformin in Wistar rats. Wistar rats were orally treated with GRT (50 mg/kg BW), atorvastatin (40 mg/kg BW) or metformin (150 mg/kg BW) alone or 50 mg/kg BW GRT in combination with 40 mg/kg BW atorvastatin or 150 mg/kg BW metformin. Blood samples were collected at 0, 10, and 30 min and 1, 2, 4, 6, and 8 h and plasma samples obtained for Liquid chromatography-mass spectrometry (LC-MS/MS) analyses. Non-compartment and two-compartment pharmacokinetic parameters of atorvastatin and metformin in the presence or absence of GRT were determined by PKSolver version 2.0 software. Membrane transporter proteins, ATP-binding cassette sub-family C member 2 (Abcc2), solute carrier organic anion transporter family, member 1b2 (Slco1b2), ATP-binding cassette, sub-family B (MDR/TAP), member 1A (Abcb1a), and organic cation transporter 1 (Oct1) mRNA expression were determined using real-time PCR expression data normalized to ß-actin and hypoxanthine-guanine phosphoribosyltransferase (HPRT), respectively. Co-administration of GRT with atorvastatin substantially increased the maximum plasma concentration (Cmax) and area of the plasma concentration-time curve (AUC0-8) of atorvastatin by 5.8-fold (p = 0.03) and 5.9-fold (p = 0.02), respectively. GRT had no effect on the plasma levels of metformin. GRT increased Abcc2 expression and metformin downregulated Abcb1a expression while the combination of GRT with atorvastatin or metformin did not significantly alter the expression of Slco1b1 or Oct1 did not significantly alter the expression of Sclo1b2 or Oct1. Co-administration of GRT with atorvastatin in rats may lead to higher plasma concentrations and, therefore, to an increase of the exposure to atorvastatin.

Polyphenolic and Physicochemical Properties of Simple-Spined Num-Num (Carissa edulis) Fruit Harvested at Ripe Stage of Maturation.

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 ± 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 ± 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.

Relationship between total dissolved solids and electrical conductivity in Marcellus hydraulic fracturing fluids.

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.

Continuous Hydrologic and Water Quality Monitoring of Vernal Ponds.

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.

Analysis of Phenolic Compounds in Rooibos Tea (Aspalathus linearis) with a Comparison of Flavonoid-Based Compounds in Natural Populations of Plants from Different Regions.

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.

A new ataxia-telangiectasia mutation in an 11-year-old female.

Ataxia-telangiectasia (A-T), a rare inherited disorder, usually affects the nervous and immune systems, and occasionally other organs. A-T is associated mainly with mutations in the ataxia telangiectasia mutated (ATM) gene, which encodes a protein kinase that has a major role in the cellular response to DNA damage. We report here a novel ATM mutation (c.3244_3245insG; p.His1082fs) in an 11-year old female. This subject presented with typical features, with the addition of chest manifestations including mediastinal lymphadenopathy and diffuse bilateral micronodular infiltration of the lungs, along with a high EBV titer. The subject died as a result of rapid B-cell lymphoma progression before chemotherapy could be initiated. This case highlights the need for the rapid diagnosis of A-T mutations and the detection of associated life-threatening outcomes such as cancers.

Expanding the ataxia with oculomotor apraxia type 4 phenotype.

Ataxia with oculomotor apraxia type 4 (AOA4) is an autosomal recessive (AR) disorder recently delineated in a Portuguese cohort and caused by mutations in the PNKP (polynucleotide kinase 3'-phosphatase) gene.(1) AOA4 is a progressive, complex movement disorder that includes hyperkinetic features, eye movement abnormalities, polyneuropathy, varying degrees of cognitive impairment, and obesity. PNKP mutations were initially discovered to be the cause of the severe nonprogressive syndrome microcephaly, early-onset intractable seizures, and developmental delay (MCSZ).(2) Here we describe a patient with compound heterozygous PNKP mutations presenting with an AOA4 phenotype. New features that we report include both mutations, presence of chorea, absence of oculomotor apraxia (OMA), and slow disease progression.

Longitudinal analysis of the neurological features of ataxia-telangiectasia.

AIM: To assess the relationship between genotype and neurological progression in ataxia-telangiectasia (A-T). METHODS: Clinical and laboratory data were extracted retrospectively from the records of patients attending the UK National Ataxia-Telangiectasia Clinic. Neurological assessments were performed using the A-T Index (Crawford Score) and the A-T Neurological Examination Scale Toolkit (A-T NEST). Variables influencing phenotype were identified by using an information-theoretic approach starting from a maximal model to generate estimates of coefficients for each variable. Per-individual progression was assessed for patients with three or more clinic attendances. RESULTS: The genotype could be determined for 125/135 patients. Crawford and A-T NEST scores were well correlated. For both scoring systems the estimated coefficients were significantly positive for Age x kinase activity but not Age x protein expression. Unlike the per-genotype analysis, the individual progression of neurological scores in the 34 patients that attended on three or more occasions was not smooth and linear (and in some cases improved over time). INTERPRETATION: Residual kinase activity confers a milder phenotype but there is no difference between kinase-dead and protein-null genotypes. The non-linear progression of individual patients' neurological scores may reflect biological complexity, day-to-day variability, limitations of the assessment methods or a combination of all three.

ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective chronic lymphocytic leukemia cells.

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.