Identification of Substituted Amino Acid Hydrazides as Novel Anti-Tubercular Agents, Using a Scaffold Hopping Approach.

Discovery and development of new therapeutic options for the treatment of Mycobacterium tuberculosis (Mtb) infection, particularly drug-resistant strains, are urgently required to tackle the global burden of this disease. Herein, we reported the synthesis of a novel series of N-substituted amino acid hydrazides, utilising a scaffold hopping approach within a library of anti-tubercular agents. Efficacy and selectivity were evaluated against three strains of Mtb (wild-type, isoniazid-resistant and rifampicin-resistant), and cytotoxicity against macrophages in vitro. The antibacterial activity and therapeutic index of these molecules were significantly affected by modifications with the N-substituents. Introduction of a 3,5-dinitroaryl moiety demonstrated enhanced antibacterial activity against all three strains of Mtb. In contrast, the inclusion of an imidazo [1,2-a]pyridine-3-carboxy moiety resulted in enhanced activity towards isoniazid mono-resistant Mtb relative to wild-type Mtb. Consequently, this scaffold hopping approach showed significant promise for exemplification of novel molecules with specific activity profiles against drug-resistant tuberculosis.

Identification of Novel Benzoxa-[2,1,3]-diazole Substituted Amino Acid Hydrazides as Potential Anti-Tubercular Agents.

Discovery and development of new therapeutic options for the treatment of Mycobacterium tuberculosis (Mtb) infection are desperately needed to tackle the continuing global burden of this disease and the efficacy and cost limitations associated with current medicines. Herein, we report the synthesis of a series of novel benzoxa-[2,1,3]-diazole substituted amino acid hydrazides in a two-step synthesis and evaluate their inhibitory activity against Mtb and selected bacterial strains of clinical importance utilising an end point-determined REMA assay. Alongside this, their potential for undesired cytotoxicity against mammalian cells was assessed employing standard MTT assay methodologies. It has been demonstrated using modification at three sites (the hydrazine, amino acid, and the benzodiazole) it is possible to change both the antibacterial activity and cytotoxicity of these molecules whilst not affecting their microbial selectivity, making them attractive architectures for further exploitation as novel antibacterial agents.

Quantitation of glyphosate, glufosinate, and AMPA in drinking water and surface waters using direct injection and charged-surface ultra-high performance liquid chromatography-tandem mass spectrometry.

Herbicides glyphosate (N-(phosphonomethyl)glycine) and glufosinate (2-amino-4-(hydroxymethylphosphinyl)butanoic acid) and the main transformation product of glyphosate, aminomethanephosphonic acid (AMPA), are challenging to analyze for in environmental samples. The quantitative method developed by this study adapts previously standardized dechlorination procedures coupled to a novel charged surface C18 column, ultra-high performance liquid chromatography-tandem mass spectrometry, polarity switching, and direct injection. The method was applied to chlorinated tap water, as well as river samples, collected in the City of Winnipeg and rural Manitoba, Canada. Using only syringe filtration without derivatization, the validated method resulted in good accuracies in both tap and surface water, at both 2 and 20 µg L-1. Method limits of detection (MLD) and quantification (MLQ) ranged from 0.022/0.074 to 0.11/0.36 µg L-1, with precisions of 0.46-2.2% (intraday) and 1.3-7.3% (interday). The mean (SEM) of the pesticides in µg L-1 for tap water were 0.11 (0.007) (AMPA), glufosinate and glyphosate < MLDs; and for Red River water were 0.56 (0.045) (AMPA), glufosinate < MLQ, and glyphosate 0.40 (0.072). For the smaller tributaries, glufosinate was >MLD but < MLQ once and that was for Shannon Creek at 0.2 µg L-1. For the remaining rivers, the mean concentrations ranged from 0.31 to 3.1 µg L-1 for AMPA, and 0.087-0.53 µg L-1 for glyphosate. The method will be ideal for supporting monitoring and risk assessment programs that require high throughput sampling and quantitative methods capable of producing robust results that leverages chromatographic and mass spectrometric paradigms instead of being extraction technology focused.

Quantitation of Canadian organic ultraviolet filters using polarity switching and ultra-high performance liquid chromatography-tandem mass spectrometry.

Ultraviolet filters (UVFs) absorb UV light and are comprised of numerous classes of compounds including inorganic and organic. They have been used for decades in protecting humans from skin damage and cancer. Recent studies have shown that UVFs are found in many phases of abiotic and biotic systems with their physical-chemical characteristics determining environmental fate and potential biological impacts such as bioaccumulation. This study developed a unified method to quantify eight UVFs (avobenzone, dioxybenzone, homosalate, octinoxate, octisalate, octocrylene, oxybenzone, and sulisobenzone) by solid phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry using polarity switching. The validated method resulted in accuracies ranging from 75 to 112%, MLD/MLQs of 0.00015/ 0.00049 to 0.0020/ 0.0067 ng mL-1, and precisions of 1.8 to 22.6% (intraday) and 1.3 to 17.2% (interday). The method was applied to chlorinated outdoor pool waters in the City of Winnipeg, Manitoba, Canada. This method could be adapted for a variety of chlorinated and unchlorinated waters such as drinking water, wastewater, and surface waters.

Characterization of a beta-hydroxybutyryl-CoA dehydrogenase from Mycobacterium tuberculosis.

The lipid-rich cell wall of mycobacteria is essential not only for virulence but also for survival. Whilst anabolic pathways for mycobacterial lipid biosynthesis have been well studied, there has been little research looking into lipid catabolism. The genome of Mycobacterium tuberculosis encodes multiple enzymes with putative roles in the beta-oxidation of fatty acids. In this report we explore the functionality of FadB2, one of five M. tuberculosis homologues of a beta-hydroxybutyryl-CoA dehydrogenase, an enzyme that catalyses the third step in the beta-oxidation cycle. Purified M. tuberculosis FadB2 catalysed the in vitro NAD(+)-dependent dehydration of beta-hydroxybutyryl-CoA to acetoacetyl-CoA at pH 10. Mutation of the active-site serine-122 residue resulted in loss of enzyme activity, consistent with the function of FadB2 as a fatty acyl dehydrogenase involved in the beta-oxidation of fatty acids. Surprisingly, purified FadB2 also catalysed the reverse reaction, converting acetoacetyl-CoA to beta-hydroxybutyryl-CoA, albeit in a lower pH range of 5.5-6.5. Additionally, a null mutant of fadB2 was generated in Mycobacterium smegmatis. However, the mutant showed no significant differences from the wild-type strain with regard to lipid composition, utilization of different fatty acid carbon sources and tolerance to various stresses; the absence of any phenotype in the mutant strain could be due to the potential redundancy between the five M. smegmatis fadB paralogues.

Insitu kinetics of human pharmaceutical conjugates and the impact of transformation, deconjugation, and sorption on persistence in wastewater batch bioreactors.

The fate of selected common pharmaceuticals and four of their major conjugates in wastewater batch bioreactors was evaluated to determine how treatment plant parameters such as addition of air, and the presence of waste activated sludge (WAS) could influence the removal of parent compounds and conjugates. Under a realistic hydraulic residence time (HRT) for each treatment sub-process of approximately 2 h, acetaminophen and its sulfate metabolite were both rapidly degraded (>99%). Propranolol was sulfated and concurrently removed. Deconjugation of N-acetylsulfamethoxazole and sulfamethoxazole-glucuronide contributed to increases of the parent sulfamethoxazole. Thyroxine was resistant to degradation, while thyroxine-glucuronide was rapidly deconjugated (>90% in <2 h). In the absence of WAS, sorption to suspended solids was another major removal mechanism for acetaminophen, propranolol, sulfamethoxazole, and thyroxine. However, with WAS, concentrations associated with suspended solids decreased for all analytes within 24 h. These results indicate that both conjugation and back-transformation are compound-specific and dependent on parameters such as HRT, addition of microbial content, and suspended solids levels. Therefore, conjugation-deconjugation processes may strongly influence the speciation of pharmaceuticals and their fate in wastewater treatment plant effluents.

Loss of a mycobacterial gene encoding a reductase leads to an altered cell wall containing beta-oxo-mycolic acid analogs and accumulation of ketones.

Mycolic acids are essential components of the mycobacterial cell wall. In this study, we show that a gene encoding a reductase involved in the final step of mycolic acid biosynthesis can be deleted in Mycobacterium smegmatis without affecting cell viability. Deletion of MSMEG4722 (ortholog of Mycobacterium tuberculosis Rv2509) altered culture characteristics and antibiotic sensitivity. The DeltaMSMEG4722 strain synthesized alpha-alkyl, beta-oxo intermediates of mycolic acids, which were found esterified to cell wall arabinogalactan. While the precursors could not be isolated directly due to their inherent instability during base treatment, their presence was established by prior reduction of the beta-oxo group by sodium borohydride. Interestingly, the mutant also accumulated unsaturated ketones, similar to tuberculenone from M. tuberculosis, which were shunt products derived from spontaneous decarboxylation of alpha-alkyl, beta-oxo fatty acid precursors of mycolic acids.

Crystal structure of Mycobacterium tuberculosis FadB2 implicated in mycobacterial ß-oxidation.

The intracellular pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis, which is a leading cause of mortality worldwide. The survival of M. tuberculosis in host macrophages through long-lasting periods of persistence depends, in part, on breaking down host cell lipids as a carbon source. The critical role of fatty-acid catabolism in this organism is underscored by the extensive redundancy of the genes implicated in ß-oxidation (∼100 genes). In a previous study, the enzymology of the M. tuberculosis L-3-hydroxyacyl-CoA dehydrogenase FadB2 was characterized. Here, the crystal structure of this enzyme in a ligand-free form is reported at 2.1 Šresolution. FadB2 crystallized as a dimer with three unique dimer copies per asymmetric unit. The structure of the monomer reveals a dual Rossmann-fold motif in the N-terminal domain, while the helical C-terminal domain mediates dimer formation. Comparison with the CoA- and NAD+-bound human orthologue mitochondrial hydroxyacyl-CoA dehydrogenase shows extensive conservation of the residues that mediate substrate and cofactor binding. Superposition with the multi-catalytic homologue M. tuberculosis FadB, which forms a trifunctional complex with the thiolase FadA, indicates that FadB has developed structural features that prevent its self-association as a dimer. Conversely, FadB2 is unable to substitute for FadB in the tetrameric FadA-FadB complex as it lacks the N-terminal hydratase domain of FadB. Instead, FadB2 may functionally (or physically) associate with the enoyl-CoA hydratase EchA8 and the thiolases FadA2, FadA3, FadA4 or FadA6 as suggested by interrogation of the STRING protein-network database.

Validated quantitative cannabis profiling for Canadian regulatory compliance - Cannabinoids, aflatoxins, and terpenes.

In response to the Canadian federal government's Cannabis Tracking and Licensing System compliance standards, a quantitative method was created for cannabis analysis, and validated using Eurachem V.2 (2014) guidelines. Cannabinol, cannabidiol, cannabigerol, cannabichromene, cannabidiolic acid, cannabigerolic acid, Δ-9-tetrahydrocannabinol, and Δ-9-tetrahydrocannabinolic acid A were all analysed by scheduled multiple reaction monitoring (MRM) via LC-MS/MS and isotope dilution. In addition, aflatoxins B1, B2, G1, and G2 were also analysed by scheduled MRM via LC-MS/MS and matrix matched calibration curves in order to achieve the reporting limits (≤2 µg kg-1) set out by the European Pharmacopoeia. The LODs/LOQs were 0.50/1.7, 2.0/6.7, 0.59/2.0, and 0.53/1.8 µg kg-1, for B1, B2, G1, and G2 respectively. Thirty one terpenes were analysed by selected reaction monitoring via GC-MS/MS and isotope dilution using ß-myrcene-d6 as a surrogate. All quantitative analyses can be accomplished using less than 1 g of material, with minimal solvent and consumable use, on low resolution instruments in less than 30 min of instrument time. Of important note is this method's power of selectivity, working ranges, and lack of need for extraction consumables such as SPE or QuEChERS, thereby minimising analytical costs and time.

Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY.

PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(deltaPE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipY(mar), in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipY(mar) in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipY(mar) was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(deltaPE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(deltaPE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(deltaPE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.

X-ray crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase II (mtKasB).

Mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C(56)) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis beta-ketoacyl ACP synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 A resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds, which inhibit mycobacterial KAS enzymes more effectively.

Regulation of cell wall synthesis and growth.

The replication and growth of Mycobacterium tuberculosis are fundamentally linked to the synthesis and extension of its complex cell wall. Incorporation of new wall material must be tightly regulated so that its deposition does not compromise the extant structure. M. tuberculosis also produces an impressive array of complex bioactive lipids that are intimately involved in pathogenesis and protective immunity. The profiles of these lipids are regulated appropriately to allow the bacterium to respond to the prevailing conditions it faces in vivo. A number of regulatory strategies employed by M. tuberculosis to control cell wall biosynthesis and cell division have now been elucidated. The review highlights the role of alternative sigma factors with extracytoplasmic function in the activation of genes for biosynthesis of complex lipids involved in pathogenicity. Rel(Mtb) and CRP(Mt) play roles in cell wall responses to general nutrient deprivation by synthesis and sensing of starvation second messengers, respectively. Recently, the importance of protein phosphorylation networks in cell wall biosynthesis has attracted considerable interest. A plethora of two-component and eukaryotic-like serine/threonine protein kinases systems have been discovered and several are implicated in cell-division, morphogenesis and regulation of the profile of complex bioactive lipids elaborated by the pathogen.

Distribution and fate of pharmaceuticals and their metabolite conjugates in a municipal wastewater treatment plant.

Some pharmaceutical conjugates can be excreted into wastewaters at levels rivalling those of the parent compounds; however, little is known about this potential reservoir of pharmaceuticals to aquatic systems. We evaluated the occurrence and distribution of four different classes of pharmaceuticals and their metabolite conjugates in a wastewater treatment plant over four months. Aqueous and suspended solids fractions of primary, mixed liquor, secondary, and final effluent, along with return activated sludge, and waste activated sludge were assessed. The only conjugate not found in the final effluent was acetaminophen sulfate. Moreover, thyroxine and thyroxine glucuronide were the only compounds quantified in the suspended solids in the final effluent. Propranolol, propranolol sulfate, thyroxine, and thyroxine glucuronide all had no significant decreases in concentration going through the wastewater treatment process, from primary to final effluent. However, there were significant decreases observed for acetaminophen (99.8%), sulfamethoxazole (71%), N-acetyl sulfamethoxazole (59%), and sulfamethoxazole glucuronide (79%). The mean (±SEM) mass loadings in the aqueous fraction of the final effluent for each compound ranged from 0.84 ±â€¯0.2 g/d for thyroxine to 45.3 ±â€¯4.2 g/d for acetaminophen. At least as much conjugate was released into receiving waters, if not more: 1.6 ±â€¯0.2 g/d for thyroxine glucuronide to 18.5 ±â€¯4.5 g/d for sulfamethoxazole glucuronide, and 61.2 ±â€¯9.6 g/d for N-acetyl sulfamethoxazole. Additionally, the mean loading of thyroxine was 0.29 ±â€¯0.025 g/day and thyroxine glucuronide 1.8 ±â€¯0.59 g/day in the suspended solids. This equates to 26% of total thyroxine and 53% of total thyroxine glucuronide associated with suspended particulate matter that reaches receiving waters. This study reflects the importance of including phase II conjugates in assessing overall compound load of pharmaceutical discharge from wastewaters, and also that substantial amounts of such contaminants are associated with wastewater solids when drugs are in the pg/L to µg/L range.

Removal of antibiotic sulfamethoxazole by anoxic/anaerobic/oxic granular and suspended activated sludge processes.

This study investigates the removal of the antibiotic sulfamethoxazole (SMX) in two sets of anoxic/anaerobic/oxic sequencing batch reactors inoculated with either suspended or granular activated sludge. Continuously, for three months, 2 µg/L SMX was spiked into the reactor feeds in a synthetic municipal wastewater with COD, total nitrogen (TN) and total phosphorous (TP) of 400, 43 and 7 mg/L, respectively. The presence of SMX had no significant impact on treatment performance of the suspended and granular biomass. After 12 h of hydraulic retention time, SMX removal efficiencies of 84 and 73% were obtained for the granular and suspended biomass, respectively. Mixing without aeration did not remove SMX, confirming the insignificance of SMX removal via sorption. The pseudo-first order SMX removal rate constants in the granular and suspended biomass were 2.25 ±â€¯0.30 and 1.34 ±â€¯0.39 L/gVSS·d, respectively. The results suggest that granules with advantages such as elevated biomass retention and greater biomass concentration could be effective for the removal of this class of antibiotics.

Variation in bacterial community structure of aerobic granular and suspended activated sludge in the presence of the antibiotic sulfamethoxazole.

The treatment performance and bacterial community structure of conventional activated sludge and aerobic granules exposed to antibiotic sulfamethoxazole (SMX) was studied. For three months, two sets of sequencing batch reactors inoculated with conventional and granular biomass were fed with a synthetic municipal wastewater containing 2 µg/L SMX. The presence of SMX had no significant impacts on treatment performance of the reactors as well as stability of the granules. Results confirmed different bacterial community structure of flocs and granules. During the operation, variations in bacterial community structure of suspended and granular sludge were observed in all reactors. The variations in bacterial community composition due to the exposure to 2 µg/L SMX were found after two months in both suspended and granular biomass. Nitrosomonas, Pseudomonas, and Acinetobacter were detected as the genes capable of degrading SMX in both biomass types. Also, Rikenellaceae, Oscillospira, Rhodocyclaceae, Zoogloea, and Shewanella varied in abundance over the operation time. Rikenellaceae and Oscillospira were vulnerable to SMX and decreased in abundance the operation time; while Rhodocyclaceae, Zoogloea, Shewanella, and Aeromonas were found as SMX resistance genes.

Dimerization of inositol monophosphatase Mycobacterium tuberculosis SuhB is not constitutive, but induced by binding of the activator Mg2+.

BACKGROUND: The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase). Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear. RESULTS: We determined the crystal structure, to 2.6 A resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce dimerization on its own. CONCLUSION: The correlation of Mg2+-driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB activity.

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT).

Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 A resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni2+ ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

Measurement of thyroxine and its glucuronide in municipal wastewater and solids using weak anion exchange solid phase extraction and ultrahigh performance liquid chromatography-tandem mass spectrometry.

A solids extraction method, using sonication in combination with weak anion exchange solid phase extraction, was created to extract thyroxine (T4) and thyroxine-O-ß-d-glucuronide (T4-Glc) simultaneously from wastewaters and sludges, and to quantify these compounds via reversed-phase ultra-high performance liquid chromatography-tandem mass spectrometry. The method limits of quantification were all in the low ng/g (dry weight solids) range for both T4 and T4-Glc: 2.13 and 2.63ng/g respectively in primary wastewater, 4.3 and 28.3ng/g for primary suspended solids, for 1.1 and 3.7ng/g for return activated sludge. Precision for measurements of T4 and T4-Glc were 2.6 and 6.5% (intraday) and 9.6 and 5.7% (interday) respectively, while linearity was 0.9967 and 0.9943 respectively. Overall recoveries for T4 and T4-Glc in primary suspended solids were 94% and 95%, and 86 and 101% in primary wastewater, respectively. Extraction efficiency tests using primary sludge determined that one methanol aliquot was sufficient during the extraction process as opposed to 2 or 3 aliquots. Mass loadings at the North Main Wastewater Treatment Plant in Winnipeg, Canada showed 316%, 714%, and 714% greater T4-Glc than T4 associated with the suspended solids of the primary, secondary, and final effluent respectively, yet 765% more T4 than T4-Glc associated with the solids of the mixed liquor. Moreover, 26% of T4 and 49% of T4-Glc were associated with the suspended solids during the treatment process. This method demonstrates the need to assess accurately both metabolite conjugates of contaminants of emerging concern, as well as the sorbed levels of particle-reactive analytes such as T4 in the aquatic environment.

Synthesis and SAR evaluation of novel thioridazine derivatives active against drug-resistant tuberculosis.

The neuroleptic drug thioridazine has been recently repositioned as possible anti-tubercular drug. Thioridazine showed anti-tubercular activity against drug resistant mycobacteria but it is endowed with adverse side effects. A small library of thioridazine derivatives has been designed through the replacement of the piperidine and phenothiazine moieties, with the aim to improve the anti-tubercular activity and to reduce the cytotoxic effects. Among the resulting compounds, the indole derivative 12e showed an antimycobacterial activity significantly better than thioridazine and a cytotoxicity 15-fold lower.

Simultaneous quantification of propranolol and sulfamethoxazole and major human metabolite conjugates 4-hydroxy-propranolol sulfate and sulfamethoxazole-ß-glucuronide in municipal wastewater-A framework for multiple classes of drugs and conjugates.

Recent data suggests there are non-trivial amounts of human pharmaceutical conjugates potentially entering environmental surface waters. These compounds could contribute to eliciting toxic effects on aquatic biota either directly or indirectly, via de-conjugation. The need for developing a single method for quantifying both parents and conjugates is necessary. Propranolol (PRO), sulfamethoxazole (SMX), and their respective major conjugates 4-OH-propranolol sulfate (PRO-Sul) and sulfamethoxazole-ß-glucuronide (SMX-Glc) were successfully simultaneously extracted through weak anion exchange solid phase extraction cartridges from primary and secondary clarification wastewaters from the North End Winnipeg Water Pollution Treatment Plant in Winnipeg, Canada. Subsequent separation and quantification were achieved by reversed-phase C18 chromatography coupled to positive electrospray ionisation tandem mass spectrometry. Linearity for all compounds throughout the 7-point calibration range was >0.99. Recovery RSD ranges across all matrices for PRO, SMX, PRO-Sul, and SMX-Glc were 2.1-13.2%, 2.3-10.2%, 9.8-19.2%, and 2.0-10.3% respectively. Primary and secondary filtrates respectively showed a significant increase of PRO from 0.039 to 0.045µg/L; a significant decrease for SMX from 1.56 to 0.58µg/L; significant decrease of PRO-Sul from 0.050 to 0.020µg/L; and a significant decrease of SMX-Glc from 0.41 to 0.019µg/L. These observations indicate that there was removal of all compounds, except for PRO, from the aqueous phase occurring at some point between the stages of treatment. To our knowledge, this is first study that simultaneously separated and quantified two different classes of parent compounds and two different kinds of human metabolite conjugates (glucuronide and sulfate) from a major urban wastewater treatment plant.