In vitro susceptibility of recent Chlamydia trachomatis clinical isolates to the CtHtrA inhibitor JO146

© 2015 Institut Pasteur. The present study aimed to establish if a previously identified Chlamydia trachomatis HtrA (CtHtrA) inhibitor, JO146, is effective against currently circulating clinical isolates to validate if CtHtrA is a clinically relevant target for future therapeutic development. Inhibition of CtHtrA during the middle of the chlamydial replicative cycle until the completion of the cycle resulted in loss of infectious progeny for six unique clinical isolates representing different serovars. This supports the potential for CtHtrA to be a clinically relevant target for development of new therapeutics and suggests the importance of further investigation of JO146 as a lead compound

Assay strategies for the discovery and validation of therapeutics targeting <i>Brugia pahangi</i> Hsp90

The chemotherapy of lymphatic filariasis relies upon drugs such as diethylcarbamazine and ivermectin that largely target the microfilarial stages of the parasite, necessitating continued treatment over the long reproductive life span of the adult worm. The identification of compounds that target adult worms has been a long-term goal of WHO. Here we describe a fluorescence polarization assay for the identification of compounds that target Hsp90 in adult filarial worms. The assay was originally developed to identify inhibitors of Hsp90 in tumor cells, and relies upon the ability of small molecules to inhibit the binding of fluorescently labelled geldanamycin to Hsp90. We demonstrate that the assay works well with soluble extracts of Brugia, while extracts of the free-living nematode C. elegans fail to bind the probe, in agreement with data from other experiments. The assay was validated using known inhibitors of Hsp90 that compete with geldanamycin for binding to Hsp90, including members of the synthetic purine-scaffold series of compounds. The efficacy of some of these compounds against adult worms was confirmed in vitro. Moreover, the assay is sufficiently sensitive to differentiate between binding of purine-scaffold compounds to human and Brugia Hsp90. The assay is suitable for high-throughput screening and provides the first example of a format with the potential to identify novel inhibitors of Hsp90 in filarial worms and in other parasitic species where Hsp90 may be a target

Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer

Background Endocrine therapies are the mainstay of treatment for oestrogen receptor (ER)-positive (ER+) breast cancer (BC). However, resistance remains problematic largely due to enhanced cross-talk between ER and growth factor pathways, circumventing the need for steroid hormones. Previously, we reported the anti-proliferative effect of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models; however, potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB, and mTORC1) may provide enhanced long-term clinical utility. Methods A panel of ER+ BC cell lines adapted to long-term oestrogen deprivation (LTED) and expressing ESR1wt or ESR1Y537S, modelling acquired resistance to an aromatase-inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan-ERBB inhibitor) in the presence or absence of oestradiol (E2), tamoxifen (4-OHT), or fulvestrant (ICI182780). End points included proliferation, cell signalling, cell cycle, and effect on ER-mediated transactivation. An in-vivo model of AI resistance was treated with monotherapies and combinations to assess the efficacy in delaying tumour progression. RNA-seq analysis was performed to identify changes in global gene expression as a result of the indicated therapies. Results Here, we show RAD001 and neratinib (pan-ERBB inhibitor) caused a concentration-dependent decrease in proliferation, irrespective of the ESR1 mutation status. The combination of either agent with endocrine therapy further reduced proliferation but the maximum effect was observed with a triple combination of RAD001, neratinib, and endocrine therapy. In the absence of oestrogen, RAD001 caused a reduction in ER-mediated transcription in the majority of the cell lines, which associated with a decrease in recruitment of ER to an oestrogen-response element on the TFF1 promoter. Contrastingly, neratinib increased both ER-mediated transactivation and ER recruitment, an effect reduced by the addition of RAD001. In-vivo analysis of an LTED model showed the triple combination of RAD001, neratinib, and fulvestrant was most effective at reducing tumour volume. Gene set enrichment analysis revealed that the addition of neratinib negated the epidermal growth factor (EGF)/EGF receptor feedback loops associated with RAD001. Conclusions Our data support the combination of therapies targeting ERBB2/3 and mTORC1 signalling, together with fulvestrant, in patients who relapse on endocrine therapy and retain a functional ER

Durability monitoring of long-lasting insecticidal (mosquito) nets (LLINs) in Madagascar: physical integrity and insecticidal activity

Abstract Background Long-lasting insecticidal mosquito nets (LLINs) are highly effective for malaria prevention. However, it is also clear that durability monitoring is essential to predict when, post-distribution, a net population, no longer meets minimum WHO standards and needs to be replaced. Following a national distribution campaign in 2013, we tracked two durability indicators, physical integrity and bio-efficacy at six and 12 months post-distribution. While the loss of net integrity during this period was in line with expectations for a one-year net life, bio-efficacy results suggested that nets were losing insecticidal effect faster than expected. The rate of bio-efficacy loss varied significantly between different net brands. Methods We tested 600 randomly selected LLINs, 200 from each of three net brands. Each brand came from different eco-epidemiological zones reflecting the original distribution scheme. Fabric integrity (size and number of holes) was quantified using the proportional hole index (pHI). A subsample of the nets, 134 new nets, 150 at six months and 124 at 12 months, were then tested for bio-efficacy using the World Health Organization (WHO) recommended method. Results Three net types, Netprotect®, Royalsentry® and Yorkool®, were followed. After six months, 54%, 39% and 45%, respectively, showed visible loss of integrity. The median pHI by type was estimated to be one, zero and one respectively. The percentage of damaged nets increased after 12 months such that 83.5%, 74% and 68.5%, had holes. The median pHI for each brand of nets was 47.5, 47 and 23. No significant difference in the estimated pHI at either six or 12 months was observed. There was a statistically significant difference in the proportion of hole size category between the three brands (χ 2 = 15.761, df = 4, P = 0.003). In cone bio-assays, mortality of new Yorkool® nets was surprisingly low (48.6%), mortality was 90.2% and 91.3% for Netprotect® and Royalsentry® (F (2, 131) = 81.59, P < 0.0001), respectively. At 12 month use, all tested nets were below the WHO threshold for replacement. Conclusion These findings suggest that there is a need for better net quality control before distribution. More frequent replacement of LLINs is probably not an option programmatically. Regardless of prior approval, LLIN durability monitoring for quality assessment as well as net loss following distribution is necessary to improve malaria control efforts

Imbalanced decision hierarchy in addicts emerging from drug-hijacked dopamine spiraling circuit

Despite explicitly wanting to quit, long-term addicts find themselves powerless to resist drugs, despite knowing that drug-taking may be a harmful course of action. Such inconsistency between the explicit knowledge of negative consequences and the compulsive behavioral patterns represents a cognitive/behavioral conflict that is a central characteristic of addiction. Neurobiologically, differential cue-induced activity in distinct striatal subregions, as well as the dopamine connectivity spiraling from ventral striatal regions to the dorsal regions, play critical roles in compulsive drug seeking. However, the functional mechanism that integrates these neuropharmacological observations with the above-mentioned cognitive/behavioral conflict is unknown. Here we provide a formal computational explanation for the drug-induced cognitive inconsistency that is apparent in the addicts' “self-described mistake”. We show that addictive drugs gradually produce a motivational bias toward drug-seeking at low-level habitual decision processes, despite the low abstract cognitive valuation of this behavior. This pathology emerges within the hierarchical reinforcement learning framework when chronic exposure to the drug pharmacologically produces pathologicaly persistent phasic dopamine signals. Thereby the drug hijacks the dopaminergic spirals that cascade the reinforcement signals down the ventro-dorsal cortico-striatal hierarchy. Neurobiologically, our theory accounts for rapid development of drug cue-elicited dopamine efflux in the ventral striatum and a delayed response in the dorsal striatum. Our theory also shows how this response pattern depends critically on the dopamine spiraling circuitry. Behaviorally, our framework explains gradual insensitivity of drug-seeking to drug-associated punishments, the blocking phenomenon for drug outcomes, and the persistent preference for drugs over natural rewards by addicts. The model suggests testable predictions and beyond that, sets the stage for a view of addiction as a pathology of hierarchical decision-making processes. This view is complementary to the traditional interpretation of addiction as interaction between habitual and goal-directed decision systems

DNA topoisomerases participate in fragility of the oncogene RET

Fragile site breakage was previously shown to result in rearrangement of the RET oncogene, resembling the rearrangements found in thyroid cancer. Common fragile sites are specific regions of the genome with a high susceptibility to DNA breakage under conditions that partially inhibit DNA replication, and often coincide with genes deleted, amplified, or rearranged in cancer. While a substantial amount of work has been performed investigating DNA repair and cell cycle checkpoint proteins vital for maintaining stability at fragile sites, little is known about the initial events leading to DNA breakage at these sites. The purpose of this study was to investigate these initial events through the detection of aphidicolin (APH)-induced DNA breakage within the RET oncogene, in which 144 APHinduced DNA breakpoints were mapped on the nucleotide level in human thyroid cells within intron 11 of RET, the breakpoint cluster region found in patients. These breakpoints were located at or near DNA topoisomerase I and/or II predicted cleavage sites, as well as at DNA secondary structural features recognized and preferentially cleaved by DNA topoisomerases I and II. Co-treatment of thyroid cells with APH and the topoisomerase catalytic inhibitors, betulinic acid and merbarone, significantly decreased APH-induced fragile site breakage within RET intron 11 and within the common fragile site FRA3B. These data demonstrate that DNA topoisomerases I and II are involved in initiating APH-induced common fragile site breakage at RET, and may engage the recognition of DNA secondary structures formed during perturbed DNA replication

Low-Dose Nitric Oxide as Targeted Anti-biofilm Adjunctive Therapy to Treat Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis

© 2017 The Authors Despite aggressive antibiotic therapy, bronchopulmonary colonization by Pseudomonas aeruginosa causes persistent morbidity and mortality in cystic fibrosis (CF). Chronic P. aeruginosa infection in the CF lung is associated with structured, antibiotic-tolerant bacterial aggregates known as biofilms. We have demonstrated the effects of non-bactericidal, low-dose nitric oxide (NO), a signaling molecule that induces biofilm dispersal, as a novel adjunctive therapy for P. aeruginosa biofilm infection in CF in an ex vivo model and a proof-of-concept double-blind clinical trial. Submicromolar NO concentrations alone caused disruption of biofilms within ex vivo CF sputum and a statistically significant decrease in ex vivo biofilm tolerance to tobramycin and tobramycin combined with ceftazidime. In the 12-patient randomized clinical trial, 10 ppm NO inhalation caused significant reduction in P. aeruginosa biofilm aggregates compared with placebo across 7 days of treatment. Our results suggest a benefit of using low-dose NO as adjunctive therapy to enhance the efficacy of antibiotics used to treat acute P. aeruginosa exacerbations in CF. Strategies to induce the disruption of biofilms have the potential to overcome biofilm-associated antibiotic tolerance in CF and other biofilm-related diseases

Service use of older people who participate in primary care health promotion: a latent class analysis

Background: Recruiting patients to health promotion programmes who will benefit is crucial to success. A key policy driver for health promotion in older people is to reduce health and social care use. Our aim was to describe service use among older people taking part in the Multi-dimensional Risk Appraisal for Older people primary care health promotion programme. Methods: A random sample of 1 in 3 older people (≥65 years old) was invited to participate in the Multi-dimensional Risk Appraisal for Older people project across five general practices in London and Hertfordshire. Data collected included socio-demographic characteristics, well-being and functional ability, lifestyle factors and service use. Latent class analysis (LCA) was used to identify groups based on use of the following: secondary health care, primary health care, community health care, paid care, unpaid care, leisure and local authority resources. Differences in group characteristics were assessed using univariate logistic regression, weighted by probability of class assignation and clustered by GP practice. Results: Response rate was 34% (526/1550) with 447 participants presenting sufficient data for analysis. LCA using three groups gave the most meaningful interpretation and best model fit. About a third (active well) were fit and active with low service use. Just under a third (high NHS users) had high impairments with high primary, secondary and community health care contact, but low non-health services use. Just over a third (community service users) with high impairments used community health and other services without much hospital use. Conclusion: Older people taking part in the Multi-dimensional Risk Appraisal for Older people primary care health promotion can be described as three groups: active well, high NHS users, and community service users

The global-scale impacts of climate change on water resources and flooding under new climate and socio-economic scenarios

This paper presents a preliminary assessment of the relative effects of rate of climate change (four Representative Concentration Pathways - RCPs), assumed future population (five Shared Socio-economic Pathways - SSPs), and pattern of climate change (19 CMIP5 climate models) on regional and global exposure to water resources stress and river flooding. Uncertainty in projected future impacts of climate change on exposure to water stress and river flooding is dominated by uncertainty in the projected spatial and seasonal pattern of change in climate. There is little clear difference in impact between RCP2.6, RCP4.5 and RCP6.0 in 2050, and between RCP4.5 and RCP6.0 in 2080. Impacts under RCP8.5 are greater than under the other RCPs in 2050 and 2080. For a given RCP, there is a difference in the absolute numbers of people exposed to increased water resources stress or increased river flood frequency between the five SSPs. With the ‘middle-of-the-road’ SSP2, climate change by 2050 would increase exposure to water resources stress for between approximately 920 and 3400 million people under the highest RCP, and increase exposure to river flood risk for between 100 and 580 million people. Under RCP2.6, exposure to increased water scarcity would be reduced in 2050 by 22-24%, compared to impacts under the RCP8.5, and exposure to increased flood frequency would be reduced by around 16%. The implications of climate change for actual future losses and adaptation depend not only on the numbers of people exposed to changes in risk, but also on the qualitative characteristics of future worlds as described in the different SSPs. The difference in ‘actual’ impact between SSPs will therefore be greater than the differences in numbers of people exposed to impact