Tumor-targeting bacteria as immune stimulants - the future of cancer immunotherapy?

Cancer immunotherapies have been widely hailed as a breakthrough for cancer treatment in the last decade, epitomized by the unprecedented results observed with checkpoint blockade. Even so, only a minority of patients currently achieve durable remissions. In general, responsive patients appear to have either a high number of tumor neoantigens, a preexisting immune cell infiltrate in the tumor microenvironment, or an 'immune-active' transcriptional profile, determined in part by the presence of a type I interferon gene signature. These observations suggest that the therapeutic efficacy of immunotherapy can be enhanced through strategies that release tumor neoantigens and/or produce a pro-inflammatory tumor microenvironment. In principle, exogenous tumor-targeting bacteria offer a unique solution for improving responsiveness to immunotherapy. This review discusses how tumor-selective bacterial infection can modulate the immunological microenvironment of the tumor and the potential for combination with cancer immunotherapy strategies to further increase therapeutic efficacy. In addition, we provide a perspective on the clinical translation of replicating bacterial therapies, with a focus on the challenges that must be resolved to ensure a successful outcome.

Structural Evaluation of a Nitroreductase Engineered for Improved Activation of the 5-Nitroimidazole PET Probe SN33623.

Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment.

Metabolomics identifies shared lipid pathways in independent amyotrophic lateral sclerosis cohorts.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease lacking effective treatments. This is due, in part, to a complex and incompletely understood pathophysiology. To shed light, we conducted untargeted metabolomics on plasma from two independent cross-sectional ALS cohorts versus control participants to identify recurrent dysregulated metabolic pathways. Untargeted metabolomics was performed on plasma from two ALS cohorts (cohort 1, n = 125; cohort 2, n = 225) and healthy controls (cohort 1, n = 71; cohort 2, n = 104). Individual differential metabolites in ALS cases versus controls were assessed by Wilcoxon, adjusted logistic regression and partial least squares-discriminant analysis, while group lasso explored sub-pathway level differences. Adjustment parameters included age, sex and body mass index. Metabolomics pathway enrichment analysis was performed on metabolites selected using the above methods. Additionally, we conducted a sex sensitivity analysis due to sex imbalance in the cohort 2 control arm. Finally, a data-driven approach, differential network enrichment analysis (DNEA), was performed on a combined dataset to further identify important ALS metabolic pathways. Cohort 2 ALS participants were slightly older than the controls (64.0 versus 62.0 years, P = 0.009). Cohort 2 controls were over-represented in females (68%, P < 0.001). The most concordant cohort 1 and 2 pathways centred heavily on lipid sub-pathways, including complex and signalling lipid species and metabolic intermediates. There were differences in sub-pathways that were enriched in ALS females versus males, including in lipid sub-pathways. Finally, DNEA of the merged metabolite dataset of both ALS and control cohorts identified nine significant subnetworks; three centred on lipids and two encompassed a range of sub-pathways. In our analysis, we saw consistent and important shared metabolic sub-pathways in both ALS cohorts, particularly in lipids, further supporting their importance as ALS pathomechanisms and therapeutics targets.

Prevalence of serum immunoglobulin E against cross-reactive carbohydrate determinants in allergic and nonallergic horses, and its impact on polysensitisation in serum allergen tests.

BACKGROUND: The existence of antibodies against cross-reactive carbohydrate determinants (CCDs) has been studied extensively in humans, and more recently, in dogs and cats. These antibodies can reduce the specificity of in vitro serum allergen tests. OBJECTIVES: To investigate the prevalence of anti-CCD immunoglobulin (Ig)E in both allergic and nonallergic horses as well as evaluate its potential impact on serum allergen testing. ANIMALS: Twenty-one allergic and 21 nonallergic horses. METHODS AND MATERIALS: Sera were analysed for anti-CCD IgE utilising a commercial CHO enzyme-linked immunosorbent assay (ELISA). An allergen specific Fc-ε receptor ELISA then was performed to evaluate polysensitisation, both with and without the addition of a proprietary anti-CCD blocking solution. RESULTS: Antibodies against CCD were detected in 30 of 42 horses. There was no statistically significant difference (p = 0.18) between the allergic and healthy groups in regard to anti-CCD prevalence. Horses with anti-CCD IgE exhibited more polysensitisation on serum allergen tests than horses without anti-CCD IgE in all allergen groups except mites. Polysensitisation was statistically significant at the 95% confidence interval for grasses (p <0.03), weeds (p = 0.02) and stinging insects (p = 0.0005). This was found to be true across both study groups. Inhibition with an anti-CCD blocking solution resulted in a 43% average reduction in polysensitisation. CONCLUSION AND CLINICAL IMPORTANCE: The prevalence of anti-CCD IgE of horses in this study coincides with the prevalence detected in pollen-sensitised people. Horses with anti-CCD IgE exhibited more positive reactions on serum allergen tests. By minimising potential artifactual polysensitisation, inclusion of an anti-CCD blocker may facilitate identification of allergen-specific IgE.

Contexte - L'existence d'anticorps contre les déterminants glucidiques à réaction croisée (CCD) a été largement étudiée chez l'homme et, plus récemment, chez le chien et le chat. Ces anticorps peuvent réduire la spécificité des tests d'allergènes sériques in vitro. Objectifs - Étudier la prévalence de l'immunoglobuline anti-CCD (Ig)E chez les chevaux allergiques et non allergiques et évaluer son impact potentiel sur les tests d'allergènes sériques. Animaux - Vingt et un chevaux allergiques et 21 chevaux non allergiques. Matériels et méthodes - Les sera ont été analysés pour les IgE anti-CCD en utilisant un dosage immuno-enzymatique CHO commercial (ELISA). Un ELISA du récepteur Fc-ε spécifique à l'allergène a ensuite été réalisé pour évaluer la polysensibilisation, avec et sans l'ajout d'une solution exclusive de blocage anti-CCD. Résultats - Des anticorps anti-CCD ont été détectés chez 30 des 42 chevaux. Il n'y avait pas de différence statistiquement significative (P = 0,18) entre les groupes allergiques et sains en ce qui concerne la prévalence anti-CCD. Les chevaux avec des IgE anti-CCD ont montré plus de polysensibilisation aux tests d'allergènes sériques que les chevaux sans IgE anti-CCD dans tous les groupes d'allergènes à l'exception des acariens. La polysensibilisation était statistiquement significative à l'intervalle de confiance de 95 % pour les graminées (P < 0,03), les herbacées (P = 0,02) et les insectes piqueurs (P = 0,0005). Cela s'est avéré vrai dans les deux groupes d'étude. L'inhibition avec une solution de blocage anti-CCD a entraîné une réduction moyenne de 43 % de la polysensibilisation. Conclusion et importance clinique - La prévalence des IgE anti-CCD des chevaux dans cette étude coïncide avec la prévalence détectée chez les personnes sensibilisées au pollen. Les chevaux avec des IgE anti-CCD ont présenté des réactions plus positives aux tests d'allergènes sériques. En minimisant la polysensibilisation artificielle potentielle, l'inclusion d'un bloqueur anti-CCD peut faciliter l'identification des IgE spécifiques de l'allergène.

Introducción- la existencia de anticuerpos contra los determinantes de carbohidratos de reacción cruzada (CCD) se ha estudiado ampliamente en humanos y, más recientemente, en perros y gatos. Estos anticuerpos pueden reducir la especificidad de las pruebas de alérgenos séricos in vitro. Objetivos - Investigar la prevalencia de inmunoglobulina (Ig)E anti-CCD tanto en caballos alérgicos como no alérgicos, así como evaluar su impacto potencial en las pruebas de alérgenos séricos. Animales- veintiún caballos alérgicos y 21 no alérgicos. Métodos y materiales- los sueros se analizaron para detectar IgE anti-CCD utilizando un ensayo comercial inmunoabsorbente ligado a enzimas (ELISA) para CHO. A continuación, se realizó un ELISA del receptor Fc-ε específico del alérgeno para evaluar la polisensibilización, con y sin la adición de una solución de bloqueo anti-CCD patentada. Resultados- se detectaron anticuerpos contra CCD en 30 de 42 caballos. No hubo diferencia estadísticamente significativa (P = 0,18) entre los grupos alérgicos y sanos con respecto a la prevalencia de anti-CCD. Los caballos con IgE anti-CCD exhibieron más polisensibilización en las pruebas de alérgenos séricos que los caballos sin IgE anti-CCD en todos los grupos de alérgenos excepto los ácaros. La polisensibilización fue estadísticamente significativa en el intervalo de confianza del 95 % para pastos (P < 0,03), malas hierbas (P = 0,02) e insectos picadores (P = 0,0005). Se encontró que esto fue similar en ambos grupos de estudio. La inhibición con una solución de bloqueo anti-CCD resultó en una reducción promedio del 43 % en la polisensibilización. Conclusión e importancia clínica - La prevalencia de IgE anti-CCD de los caballos en este estudio coincide con la prevalencia detectada en personas sensibilizadas al polen. Los caballos con IgE anti-CCD exhibieron más reacciones positivas en las pruebas de alérgenos en suero. La inclusión de un bloqueador anti-CCD puede facilitar la identificación de IgE específica de alérgeno al minimizar la posible polisensibilización artificial.

Contexto - A existência de anticorpos contra determinantes de carboidratos de reação cruzada (CCDs) tem sido estudada extensivamente em humanos, e, mas recentemente, em cães e gatos. Estes anticorpos podem reduzir a especificidade de testes alérgicos sorológicos in vitro. Objetivos - Investigar a prevalência de imunoglobulina (Ig)E tanto em cavalos alérgicos quanto em cavalos não alérgicos e avaliar o seu potencial impacto no teste alérgico sorológico. Animais - Vinte e um cavalos alérgicos e 21 não alérgicos. Métodos e materiais - O soro foi testado para IgE anti-CCD utilizando um ensaio imunoenzimático CHO comercial (ELISA). Um ELISA alérgeno-específico para receptor Fc-ε foi realizado para avaliar polissensibilização, com e sem a adição de uma solução de bloqueio anti-CCD. Resultados - Anticorpos anti-CCD foram encontrados em 30 de 42 cavalos. Não houve diferença estatística significativa (P = 0,18) entre os grupos alérgico e saudável quanto à prevalência de anti-CCD. Cavalos com IgE anti-CCD exibiram mais polissensibilização no teste alérgico sorológico que cavalos sem IgE anti-CCD em todos os grupos alergênicos, exceto ácaros. Polissensibilização foi estatisticamente significativa em um intervalo de confiança de 95% para gramíneas (P < 0,03), herbáceas (P = 0,02) e insetos que picam (P = 0,0005). Isto foi verdadeiro para os dois grupos estudados. Inibição com uma solução bloqueadora de anti-CCD resultou em uma redução média de 43% na polissensibilização. Conclusão e importância clínica - A prevalência de IgE anti-CCD em cavalos nesse estudo coincide com a prevalência detectada em pessoas sensibilizadas a pólen. Equinos com IgE anti-CCD apresentaram mais reações positivas nos testes alérgicos sorológicos. Minimizando uma potencial polissensibilização a partir da utilização de um bloqueador de anti-CCD pode facilitar a identificação de IgE alérgeno-específica.

An industrial perspective on co-crystals: Screening, identification and development of the less utilised solid form in drug discovery and development.

Active pharmaceutical ingredients are commonly marketed as a solid form due to ease of transport, storage and administration. In the design of a drug formulation, the selection of the solid form is incredibly important and is traditionally based on what polymorphs, hydrates or salts are available for that compound. Co-crystals, another potential solid form available, are currently not as readily considered as a viable solid form for the development process. Even though co-crystals are gaining an ever-increasing level of interest within the pharmaceutical community, their acceptance and application is still not as standard as other solid forms such as the ubiquitous pharmaceutical salt and stabilised amorphous formulations. Presented in this chapter is information that would allow for a co-crystal screen to be planned and conducted as well as scaled up using solution and mechanochemistry based methods commonly employed in both the literature and industry. Also presented are methods for identifying the formation of a co-crystal using a variety of analytical techniques as well as the importance of confirming the formation of co-crystals from a legal perspective and demonstrating the legal precedent by looking at co-crystalline products already on the market. The benefits of co-crystals have been well established, and presented in this chapter are a selection of examples which best exemplify their potential. The goal of this chapter is to increase the understanding of co-crystals and how they may be successfully exploited in early stage development.

Directed evolution of the B. subtilis nitroreductase YfkO improves activation of the PET-capable probe SN33623 and CB1954 prodrug.

OBJECTIVES: To use directed evolution to improve YfkO-mediated reduction of the 5-nitroimidazole PET-capable probe SN33623 without impairing conversion of the anti-cancer prodrug CB1954. RESULTS: Two iterations of error-prone PCR, purifying selection, and FACS sorting in a DNA damage quantifying GFP reporter strain were used to identify three YfkO variants able to sensitize E. coli host cells to at least 2.4-fold lower concentrations of SN33623 than the native enzyme. Two of these variants were able to be purified in a functional form, and in vitro assays revealed these were twofold and fourfold improved in kcat/KM with SN33623 over wild type YfkO. Serendipitously, the more-active variant was also nearly fourfold improved in kcat/KM versus wild type YfkO in converting CB1954 to a genotoxic drug. CONCLUSIONS: The enhanced activation of the PET imaging probe SN33623 and CB1954 prodrug exhibited by the lead evolved variant of YfkO offers prospects for improved enzyme-prodrug therapy.

Design, Synthesis and In-Vitro Biological Evaluation of Antofine and Tylophorine Prodrugs as Hypoxia-Targeted Anticancer Agents.

Phenanthroindolizidines, such as antofine and tylophorine, are a family of natural alkaloids isolated from different species of Asclepiadaceas. They are characterized by interesting biological activities, such as pronounced cytotoxicity against different human cancerous cell lines, including multidrug-resistant examples. Nonetheless, these derivatives are associated with severe neurotoxicity and loss of in vivo activity due to the highly lipophilic nature of the alkaloids. Here, we describe the development of highly polar prodrugs of antofine and tylophorine as hypoxia-targeted prodrugs. The developed quaternary ammonium salts of phenanthroindolizidines showed high chemical and metabolic stability and are predicted to have no penetration through the blood-brain barrier. The designed prodrugs displayed decreased cytotoxicity when tested under normoxic conditions. However, their cytotoxic activity considerably increased when tested under hypoxic conditions.

Untargeted metabolomics yields insight into ALS disease mechanisms.

OBJECTIVE: To identify dysregulated metabolic pathways in amyotrophic lateral sclerosis (ALS) versus control participants through untargeted metabolomics. METHODS: Untargeted metabolomics was performed on plasma from ALS participants (n=125) around 6.8 months after diagnosis and healthy controls (n=71). Individual differential metabolites in ALS cases versus controls were assessed by Wilcoxon rank-sum tests, adjusted logistic regression and partial least squares-discriminant analysis (PLS-DA), while group lasso explored sub-pathway-level differences. Adjustment parameters included sex, age and body mass index (BMI). Metabolomics pathway enrichment analysis was performed on metabolites selected by the above methods. Finally, machine learning classification algorithms applied to group lasso-selected metabolites were evaluated for classifying case status. RESULTS: There were no group differences in sex, age and BMI. Significant metabolites selected were 303 by Wilcoxon, 300 by logistic regression, 295 by PLS-DA and 259 by group lasso, corresponding to 11, 13, 12 and 22 enriched sub-pathways, respectively. 'Benzoate metabolism', 'ceramides', 'creatine metabolism', 'fatty acid metabolism (acyl carnitine, polyunsaturated)' and 'hexosylceramides' sub-pathways were enriched by all methods, and 'sphingomyelins' by all but Wilcoxon, indicating these pathways significantly associate with ALS. Finally, machine learning prediction of ALS cases using group lasso-selected metabolites achieved the best performance by regularised logistic regression with elastic net regularisation, with an area under the curve of 0.98 and specificity of 83%. CONCLUSION: In our analysis, ALS led to significant metabolic pathway alterations, which had correlations to known ALS pathomechanisms in the basic and clinical literature, and may represent important targets for future ALS therapeutics.

Alanine scan-guided synthesis and biological evaluation of analogues of culicinin D, a potent anticancer peptaibol.

Culicinin D (1), a 10 amino acid peptaibol originally isolated from Culicinomyces clavisporus, exhibits potent activity against a range of cancer cell lines. Building on our previous work exploring the structure-activity relationship (SAR) of the unusual (2S,4S,6R)-AHMOD residue, a series of analogues of culicinin D were prepared to further investigate the SAR of these peptaibols. Alanine scanning of a potent and readily accessible analogue 23 revealed the effect of each residue on antiproliferative activity, and a small panel of analogues were prepared to explore the SAR of the non-natural amino acid residue (2S,4R)-AMD. Results from the alanine scan were used to design an expanded library of culicinin D analogues, leading to the discovery of cyclohexylalanine analogue 52, which exhibited better antiproliferative activity than the natural product 1.

Synthesis and antiproliferative activity of C- and N-terminal analogues of culicinin D.

Culicinin D (1), a 10 amino acid peptaibol containing several unusual residues, has been shown to exhibit potent anticancer activity. Previous work in our group towards developing a structure-activity relationship (SAR) for this peptaibol has concentrated on replacement of the synthetically challenging AHMOD (3) and AMD (4) residues, resulting in the discovery of analogues with equivalent or better potency and simplified synthesis. The SAR of this peptaibol is extended in this work by investigating the effect of the N-terminal lipid tail and C-terminal amino alcohol, revealing the key contribution of each of these moieties on antiproliferative activity in a panel of breast and lung cancer cell lines.

Exploration of anomalous perceptual experiences in migraine between attacks using the Cardiff Anomalous Perceptions Scale.

Distortions in sensory experiences that precede a migraine attack have been extensively documented, the most well-known being the visual aura. Distortions in the experience of other senses are also reported as part of an aura, albeit less frequently, together with changes in the perception or ownership of the body or body parts. There are many examples of differences in aspects of visual perception between migraine and control groups, between attacks, but not as much on unusual experiences involving other senses, the sense of the body or the experience of the environment. Seventy-seven migraine (33 with aura) and 74 control participants took part. Anomalous perceptions were experienced by both migraine and control groups, but more with migraine experienced them and rated them as more distressing, intrusive and frequent. Associations with reports of visual triggers of migraine and visual discomfort are presented. This study is the first to show relationships between these factors.

Subcellular Location of Tirapazamine Reduction Dramatically Affects Aerobic but Not Anoxic Cytotoxicity.

Hypoxia is an adverse prognostic feature of solid cancers that may be overcome with hypoxia-activated prodrugs (HAPs). Tirapazamine (TPZ) is a HAP which has undergone extensive clinical evaluation in this context and stimulated development of optimized analogues. However the subcellular localization of the oxidoreductases responsible for mediating TPZ-dependent DNA damage remains unclear. Some studies conclude only nuclear-localized oxidoreductases can give rise to radical-mediated DNA damage and thus cytotoxicity, whereas others identify a broader role for endoplasmic reticulum and cytosolic oxidoreductases, indicating the subcellular location of TPZ radical formation is not a critical requirement for DNA damage. To explore this question in intact cells we engineered MDA-231 breast cancer cells to express the TPZ reductase human NADPH: cytochrome P450 oxidoreductase (POR) harboring various subcellular localization sequences to guide this flavoenzyme to the nucleus, endoplasmic reticulum, cytosol or inner surface of the plasma membrane. We show that all POR variants are functional, with differences in rates of metabolism reflecting enzyme expression levels rather than intracellular TPZ concentration gradients. Under anoxic conditions, POR expression in all subcellular compartments increased the sensitivity of the cells to TPZ, but with a fall in cytotoxicity per unit of metabolism (termed 'metabolic efficiency') when POR is expressed further from the nucleus. However, under aerobic conditions a much larger increase in cytotoxicity was observed when POR was directed to the nucleus, indicating very high metabolic efficiency. Consequently, nuclear metabolism results in collapse of hypoxic selectivity of TPZ, which was further magnified to the point of reversing O2 dependence (oxic > hypoxic sensitivity) by employing a DNA-affinic TPZ analogue. This aerobic hypersensitivity phenotype was partially rescued by cellular copper depletion, suggesting the possible involvement of Fenton-like chemistry in generating short-range effects mediated by the hydroxyl radical. In addition, the data suggest that under aerobic conditions reoxidation strictly limits the TPZ radical diffusion range resulting in site-specific cytotoxicity. Collectively these novel findings challenge the purported role of intra-nuclear reductases in orchestrating the hypoxia selectivity of TPZ.

Engineering Escherichia coli NfsB To Activate a Hypoxia-Resistant Analogue of the PET Probe EF5 To Enable Non-Invasive Imaging during Enzyme Prodrug Therapy.

Gene-directed enzyme prodrug therapy (GDEPT) uses tumor-tropic vectors to deliver prodrug-converting enzymes such as nitroreductases specifically to the tumor environment. The nitroreductase NfsB from Escherichia coli (NfsB_Ec) has been a particular focal point for GDEPT and over the past 25 years has been the subject of several engineering studies seeking to improve catalysis of prodrug substrates. To facilitate clinical development, there is also a need to enable effective non-invasive imaging capabilities. SN33623, a 5-nitroimidazole analogue of 2-nitroimidazole hypoxia probe EF5, has potential for PET imaging exogenously delivered nitroreductases without generating confounding background due to tumor hypoxia. However, we show here that SN33623 is a poor substrate for NfsB_Ec. To address this, we used assay-guided sequence and structure analysis to identify two conserved residues that block SN33623 activation in NfsB_Ec and close homologues. Introduction of the rational substitutions F70A and F108Y into NfsB_Ec conferred high levels of SN33623 activity and enabled specific labeling of E. coli expressing the engineered enzyme. Serendipitously, the F70A and F108Y substitutions also substantially improved activity with the anticancer prodrug CB1954 and the 5-nitroimidazole antibiotic prodrug metronidazole, which is a potential biosafety agent for targeted ablation of nitroreductase-expressing vectors.

High plasma concentrations of organic pollutants negatively impact survival in amyotrophic lateral sclerosis.

OBJECTIVE: To determine whether persistent organic pollutants (POP) affect amyotrophic lateral sclerosis (ALS) survival. METHODS: ALS participants seen at the University of Michigan (Ann Arbor, MI, USA) provided plasma samples for measurement of POPs. ALS disease and clinical features were collected prospectively from the medical records. Survival models used a composite summary measure of exposure due to multiple POPs (environmental risk score or ERS). RESULTS: 167 participants (40.7% female, n=68) with ALS were recruited, of which 119 died during the study period. Median diagnostic age was 60.9 years (IQR 52.7-68.2), median time from symptom onset to diagnosis was 1.01 years (IQR 0.67-1.67), bulbar onset 28.7%, cervical onset 33.5% and lumbar onset 37.7%. Participants in the highest quartile of ERS (representing highest composite exposure), adjusting for age at diagnosis, sex and other covariates had a 2.07 times greater hazards rate of mortality (p=0.018, 95% CI 1.13 to 3.80) compared with those in the lowest quartile. Pollutants with the largest contribution to the ERS were polybrominated diphenyl ethers 154 (HR 1.53, 95% CI 0.90 to 2.61), polychlorinated biphenyls (PCB) 118 (HR 1.50, 95% CI 0.95 to 2.39), PCB 138 (HR 1.69, 95% CI 0.99 to 2.90), PCB 151 (HR 1.46, 95% CI 1.01 to 2.10), PCB 175 (HR 1.53, 95% CI 0.98 to 2.40) and p,p'-DDE (HR 1.39, 95% CI 1.07 to 1.81). CONCLUSIONS: Higher concentrations of POPs in plasma are associated with reduced ALS survival, independent of age, gender, segment of onset and other covariates. This study helps characterise and quantify the combined effects of POPs on ALS and supports the concept that environmental exposures play a role in disease pathogenesis.

Prototyping kinase inhibitor-cytotoxin anticancer mutual prodrugs activated by tumour hypoxia: A chemical proof of concept study.

Amide- and ester-linked kinase inhibitor-cytotoxin conjugates were rationally designed and synthesised as prototype hypoxia-activated anticancer mutual prodrugs. Chemical reduction of an aryl nitro trigger moiety was shown to initiate a spontaneous cyclisation/fragmentation reaction that simultaneously released the kinase inhibitor semaxanib (SU5416) and the amine- or alcohol-linked cytotoxin from the prodrugs. Preliminary cell testing and reduction potential measurements support optimisation of the compounds towards tumour-selective mutual prodrugs.

Novel association of Psychrobacter and Pseudomonas with malodour in bloodhound dogs, and the effects of a topical product composed of essential oils and plant-derived essential fatty acids in a randomized, blinded, placebo-controlled study.

BACKGROUND: The pathogenesis and treatment of cutaneous malodour in dogs have not been investigated previously. Staphylococcus and Corynebacterium spp. are associated with human axillary malodour. HYPOTHESIS: Staphylococcus and Corynebacterium spp. are associated with cutaneous malodour in dogs, and treatment with a topical essential oil-based product will improve malodour and reduce the abundance of odour-causing bacteria. ANIMALS: Twenty seven bloodhound dogs from a south Texas boarding facility were enrolled in this study. METHODS AND MATERIALS: Skin swabs were taken from the axilla and dorsum of 27 dogs at initiation of the study. Mean malodour scores were used to assign dogs to control or malodour groups. The malodourous dogs were randomly assigned to a treatment or placebo group, received four weekly topical applications of the spot-on or placebo, and samples were recollected. Next-generation sequencing (NGS) and real-time quantitative PCR (qPCR) were performed on all swabs. RESULTS: Psychrobacter and Pseudomonas spp. were significantly more abundant (P < 0.001, P = 0.006; respectively), and overall bacterial diversity was reduced (P = 0.0384) on the skin of malodourous dogs. Staphylococcus and Corynebacterium spp. were not associated with malodour. The topical essential oil-based product significantly (P = 0.0078) improved malodour in the treatment group and shifted their bacterial community structure. CONCLUSIONS AND CLINICAL IMPORTANCE: A novel association of bacterial genera with malodour in bloodhound dogs, identified by NGS, highlights future targets for odour control. The topical treatment significantly reduced malodour. The interaction between the topical treatment and cutaneous microbiota should be further investigated and may be useful in other dermatological conditions involving microbiota.

Changes in blood pressure and heart rate during sedation with ketamine in the pediatric ED.

BACKGROUND: Ketamine is commonly used in the emergency department for short, painful procedures. We describe changes in blood pressure (BP) and heart rate (HR) during procedural sedation with ketamine, as these changes have not been well described in children. METHODS: We performed a secondary analysis of a prospective, observational study involving children aged 8 to 18 years who received procedural sedation with ketamine in a pediatric emergency department. Serial vital signs and sedation scores were recorded from baseline until recovery from ketamine procedural sedation. Time of orthopedic manipulation was also recorded. Linear mixed-effect models were used to evaluate changes in systolic BP (SBP), diastolic BP (DBP), and HR using 3 sedation strata: presedation (baseline), sedated (ketamine administered and patient deeply sedated), and recovery (ketamine administered with patient minimally sedated), controlling for age and weight. RESULTS: Sixty children were enrolled; 10 were excluded due to missing manipulation time. A total of 394 observations were recorded. Mean sedated SBP, DBP, and HR were 8 mm Hg, 4 mm Hg, and 13 beats/min higher than presedation SBP (P<.001), DBP (P<.01), and HR (P<.001), respectively. Mean sedated SBP and DBP were 3 and 4 mm Hg higher than SBP (P=.006) and DBP (P<.01) during recovery. Manipulation increased mean SBP by 5 mm Hg (P<.001), mean DBP by 7 mm Hg (P<.001), and mean HR by 1 beat/min (P=.35). CONCLUSIONS: Ketamine administered during procedural sedation for painful procedures causes a statistically significant but modest increase in SBP, DBP, and HR. Orthopedic manipulation further increases BP.

Characterization of the cutaneous mycobiota in healthy and allergic cats using next generation sequencing.

BACKGROUND: Next generation sequencing (NGS) studies have demonstrated a diverse skin-associated microbiota and microbial dysbiosis associated with atopic dermatitis in people and in dogs. The skin of cats has yet to be investigated using NGS techniques. HYPOTHESIS/OBJECTIVES: We hypothesized that the fungal microbiota of healthy feline skin would be similar to that of dogs, with a predominance of environmental fungi, and that fungal dysbiosis would be present on the skin of allergic cats. ANIMALS: Eleven healthy cats and nine cats diagnosed with one or more cutaneous hypersensitivity disorders, including flea bite, food-induced and nonflea nonfood-induced hypersensitivity. METHODS: Healthy cats were sampled at twelve body sites and allergic cats at six sites. DNA was isolated and Illumina sequencing was performed targeting the internal transcribed spacer region of fungi. Sequences were processed using the bioinformatics software QIIME. RESULTS: The most abundant fungal sequences from the skin of all cats were classified as Cladosporium and Alternaria. The mucosal sites, including nostril, conjunctiva and reproductive tracts, had the fewest number of fungi, whereas the pre-aural space had the most. Allergic feline skin had significantly greater amounts of Agaricomycetes and Sordariomycetes, and significantly less Epicoccum compared to healthy feline skin. CONCLUSIONS: The skin of healthy cats appears to have a more diverse fungal microbiota compared to previous studies, and a fungal dysbiosis is noted in the skin of allergic cats. Future studies assessing the temporal stability of the skin microbiota in cats will be useful in determining whether the microbiota sequenced using NGS are colonizers or transient microbes.

Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility.

This review examines the vast catalytic and therapeutic potential offered by type I (i.e. oxygen-insensitive) nitroreductase enzymes in partnership with nitroaromatic prodrugs, with particular focus on gene-directed enzyme prodrug therapy (GDEPT; a form of cancer gene therapy). Important first indications of this potential were demonstrated over 20 years ago, for the enzyme-prodrug pairing of Escherichia coli NfsB and CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, it has become apparent that both the enzyme and the prodrug in this prototypical pairing have limitations that have impeded their clinical progression. Recently, substantial advances have been made in the biodiscovery and engineering of superior nitroreductase variants, in particular development of elegant high-throughput screening capabilities to enable optimization of desirable activities via directed evolution. These advances in enzymology have been paralleled by advances in medicinal chemistry, leading to the development of second- and third-generation nitroaromatic prodrugs that offer substantial advantages over CB1954 for nitroreductase GDEPT, including greater dose-potency and enhanced ability of the activated metabolite(s) to exhibit a local bystander effect. In addition to forging substantial progress towards future clinical trials, this research is supporting other fields, most notably the development and improvement of targeted cellular ablation capabilities in small animal models, such as zebrafish, to enable cell-specific physiology or regeneration studies.

Binding mode of the breakthrough inhibitor AZD9291 to epidermal growth factor receptor revealed.

The discovery of genetic drivers of lung cancer in patient sub-groups has led to their use as predictive biomarkers and as targets for selective drug therapy. Some of the most important lung cancer drivers are mutations in the EGFR gene, for example, the exon 19 deletions and the L858R variant that confer sensitivity to the front line drugs erlotinib and gefitinib; the acquired T790M variants confer drug resistance and a poor prognosis. A challenge then in targeting EGFR is to produce drugs that inhibit both sensitising variants and resistance variants, leaving wild type protein in healthy cells unaffected. One such agent is AstraZeneca's "breakthrough" AZD9291 molecule that shows a 200-fold selectivity for T790M/L858R over wild type EGFR. Our X-ray crystal structure reveals the binding mode of AZD9291 to the kinase domain of wild type EGFR.