Activity in vivo of anti-Trypanosoma cruzi compounds selected from a high throughput screening

Novel technologies that include recombinant pathogens and rapid detection methods are contributing to the development of drugs for neglected diseases. Recently, the results from the first high throughput screening (HTS) to test compounds for activity against Trypanosoma cruzi trypomastigote infection of host cells were reported. We have selected 23 compounds from the hits of this HTS, which were reported to have high anti-trypanosomal activity and low toxicity to host cells. These compounds were highly purified and their structures confirmed by HPLC/mass spectrometry. The compounds were tested in vitro, where about half of them confirmed the anti-T. cruzi activity reported in the HTS, with IC50 values lower than 5 μM. We have also adapted a rapid assay to test anti-T. cruzi compounds in vivo using mice infected with transgenic T. cruzi expressing luciferase as a model for acute infection. The compounds that were active in vitro were also tested in vivo using this assay, where we found two related compounds with a similar structure and low in vitro IC50 values (0.11 and 0.07 μM) that reduce T. cruzi infection in the mouse model more than 90% after five days of treatment. Our findings evidence the benefits of novel technologies, such as HTS, for the drug discovery pathway of neglected diseases, but also caution about the need to confirm the results in vitro. We also show how rapid methods of in vivo screening based in luciferase-expressing parasites can be very useful to prioritize compounds early in the chain of development.     

Putting into practice domain-linear motif interaction predictions for exploration of protein networks

PDZ domains recognise short sequence motifs at the extreme C-termini of proteins. A model based on microarray data has been recently published for predicting the binding preferences of PDZ domains to five residue long C-terminal sequences. Here we investigated the potential of this predictor for discovering novel protein interactions that involve PDZ domains. When tested on real negative data assembled from published literature, the predictor displayed a high false positive rate (FPR). We predicted and experimentally validated interactions between four PDZ domains derived from the human proteins MAGI1 and SCRIB and 19 peptides derived from human and viral C-termini of proteins. Measured binding intensities did not correlate with prediction scores, and the high FPR of the predictor was confirmed. Results indicate that limitations of the predictor may arise from an incomplete model definition and improper training of the model. Taking into account these limitations, we identified several novel putative interactions between PDZ domains of MAGI1 and SCRIB and the C-termini of the proteins FZD4, ARHGAP6, NET1, TANC1, GLUT7, MARCH3, MAS, ABC1, DLL1, TMEM215 and CYSLTR2. These proteins are localised to the membrane or suggested to act close to it and are often involved in G protein signalling. Furthermore, we showed that, while extension of minimal interacting domains or peptides toward tandem constructs or longer peptides never suppressed their ability to interact, the measured affinities and inferred specificity patterns often changed significantly. This suggests that if protein fragments interact, the full length proteins are also likely to interact, albeit possibly with altered affinities and specificities. Therefore, predictors dealing with protein fragments are promising tools for discovering protein interaction networks but their application to predict binding preferences within networks may be limited.     

Association between Oestrogens Receptor Expressions in Breast Cancer and Comorbidities: A Cross-Sectional,Population-Based Study

Background

Breast cancer with oestrogen receptor expression is common in older women. Several factors, such as age and reproductive hormone exposure, have been associated with oestrogen receptor expression in breast cancer. However, the association between comorbidities and the oestrogen receptor expression has been poorly studied. We hypothesized that there was an association between burden comorbidity and breast cancer with oestrogen receptor expression in older women.

Objective

To determine whether oestrogen receptor expression in breast cancer was associated with burden comorbidity in community-dwelling women.

Methods

A total of 1,707 women with breast cancer registered on the list of a breast cancer registry were included. The recorded data included: age, Charlson Comorbidity Index score≥1, breast cancer characteristics (coded according to the International Classification of Diseases for Oncology), and breast cancer pathological stage (the pathological-tumour-node-metastasis, Scarff Bloom Richardson, and hormonal status of oestrogen receptor, progesterone receptor, and human epidermal growth factor receptor).

Results

Breast cancer with oestrogen receptor expression was identified in 1,378 patients (80·7%). The fully-adjusted logistic regression showed that oestrogen receptor expression was associated with Charlson Comorbidity Index score≥1 (odds ratio [OR] = 1·91,95%confidence interval [CI] = [1.01–3.61], P = 0·048), progesterone receptor expression (OR = 16·64, 95%CI = [11.62–23.81], P<0·001), human epidermal growth factor receptor (OR = 0·54, 95%CI = [0.34–0.84], P = 0·007), age (OR = 1.02, 95%CI = [1.00–1.03], P = 0.008), Scarff Bloom Richardson grade II and grade III (OR = 0·21with 95%CI = [0.10–0.44] and OR = 0·06 with 95%CI = [0.03–0.12], P<0·001).

Conclusion

Our findings provide new data showing an independent positive association between burden comorbidity and breast cancer with oestrogen receptor expression. This result confirms that evaluation of oestrogen receptor expression in breast cancer should not be limited to hormonal factors stratified by age.     

Large Conductance Voltage- and Ca2+-gated Potassium (BK) Channel β4 Subunit Influences Sensitivity and Tolerance to Alcohol by Altering Its Response to Kinases

Tolerance is a well described component of alcohol abuse and addiction. The large conductance voltage- and Ca²⁺-gated potassium channel (BK) has been very useful for studying molecular tolerance. The influence of association with the β4 subunit can be observed at the level of individual channels, action potentials in brain slices, and finally, drinking behavior in the mouse. Previously, we showed that 50 mm alcohol increases both α and αβ4 BK channel open probability, but only α BK develops acute tolerance to this effect. Currently, we explore the possibility that the influence of the β4 subunit on tolerance may result from a striking effect of β4 on kinase modulation of the BK channel. We examine the influence of the β4 subunit on PKA, CaMKII, and phosphatase modulation of channel activity, and on molecular tolerance to alcohol. We record from human BK channels heterologously expressed in HEK 293 cells composed of its core subunit, α alone (Insertless), or co-expressed with the β4 BK auxiliary subunit, as well as, acutely dissociated nucleus accumbens neurons using the cell-attached patch clamp configuration. Our results indicate that BK channels are strongly modulated by activation of specific kinases (PKA and CaMKII) and phosphatases. The presence of the β4 subunit greatly influences this modulation, allowing a variety of outcomes for BK channel activity in response to acute alcohol.     

Plastidial Expression of Type II NAD(P)H Dehydrogenase Increases the Reducing State of Plastoquinones and Hydrogen Photoproduction Rate by the Indirect Pathway in Chlamydomonas reinhardtii

Biological conversion of solar energy into hydrogen is naturally realized by some microalgae species due to a coupling between the photosynthetic electron transport chain and a plastidial hydrogenase. While promising for the production of clean and sustainable hydrogen, this process requires improvement to be economically viable. Two pathways, called direct and indirect photoproduction, lead to sustained hydrogen production in sulfur-deprived Chlamydomonas reinhardtii cultures. The indirect pathway allows an efficient time-based separation of O₂ and H₂ production, thus overcoming the O₂ sensitivity of the hydrogenase, but its activity is low. With the aim of identifying the limiting step of hydrogen production, we succeeded in overexpressing the plastidial type II NAD(P)H dehydrogenase (NDA2). We report that transplastomic strains overexpressing NDA2 show an increased activity of nonphotochemical reduction of plastoquinones (PQs). While hydrogen production by the direct pathway, involving the linear electron flow from photosystem II to photosystem I, was not affected by NDA2 overexpression, the rate of hydrogen production by the indirect pathway was increased in conditions, such as nutrient limitation, where soluble electron donors are not limiting. An increased intracellular starch was observed in response to nutrient deprivation in strains overexpressing NDA2. It is concluded that activity of the indirect pathway is limited by the nonphotochemical reduction of PQs, either by the pool size of soluble electron donors or by the PQ-reducing activity of NDA2 in nutrient-limited conditions. We discuss these data in relation to limitations and biotechnological improvement of hydrogen photoproduction in microalgae.A number of microalgal and cyanobacterial species are able to convert solar energy into hydrogen by photobiological processes and are therefore considered promising organisms for developing clean and sustainable hydrogen production (Benemann, 2000; Ghirardi et al., 2000; Rupprecht et al., 2006). In microalgae, hydrogen photoproduction results from coupling the photosynthetic electron transport chain and a plastidial [FeFe] hydrogenase. Under most conditions, hydrogen photoproduction is a transient phenomenon that lasts from several seconds to a few minutes (Ghirardi et al., 2000; Melis and Happe, 2001). It has been considered a relic of evolution that may now serve, under certain environmental conditions, such as induction of photosynthesis in anoxia (Ghysels et al., 2013), as a safety valve that protects the photosynthetic electron transport chain from photodamage that results from overreduction of electron acceptors (Kessler, 1973; Tolleter et al., 2011). A major limitation to sustained hydrogen photoproduction is due to the oxygen sensitivity of the [FeFe] hydrogenase (Happe et al., 2002; Stripp et al., 2009). Melis et al. (2000) proposed an elegant way to overcome this oxygen sensitivity through a time-based separation of hydrogen and oxygen production phases occurring, for instance, in response to sulfur deficiency in a closed environment. Another limitation is related to the electron supply for the hydrogenase coming from the photosynthetic electron transport chain (Cournac et al., 2002). This limitation is partly due to the fact that other metabolic pathways, such as ferredoxin-NADP⁺ reductase and CO₂ fixation, compete with the hydrogenase for the use of reduced ferredoxin (Gaffron and Rubin, 1942; Hemschemeier et al., 2008). This is also due to upstream regulation of the electron transport chain, recently evidenced from the study of a Chlamydomonas reinhardtii mutant affected in proton gradient regulation-like1 (PGRL1)-mediated cyclic electron flow (CEF) around PSI. The strong enhancement of hydrogen production rates observed in the pgrl1 mutant was interpreted as the release of a control exerted by the transthylakoidal pH gradient on electron supply to the hydrogenase (Tolleter et al., 2011).Two pathways, direct or indirect, can supply electrons to the hydrogenase (Benemann, 2000; Melis and Happe, 2001; Chochois et al., 2009). In the direct pathway, the whole electron transport chain is engaged, with PSII supplying electrons to the plastoquinone (PQ) pool, the cytochrome b₆/f complex, and, in turn, PSI, ferredoxin, and the [FeFe] hydrogenase. Due to the high oxygen sensitivity of the [FeFe] hydrogenase and to the fact that O₂ is produced during photosynthesis at PSII, the direct pathway only operates when PSII activity is lower than mitochondrial respiration, thereby allowing anaerobiosis to be maintained. Such conditions can be obtained by decreasing PSII activity either by means of sulfur deprivation (Melis et al., 2000) or by decreasing light intensity in the photobioreactor (Degrenne et al., 2010). In the indirect pathway, reducing equivalents, stored as starch during the aerobic phase, are subsequently used to fuel hydrogen production. This implies a nonphotochemical reduction of the PQ pool that is at least in part mediated by NDA2, a type II NADH dehydrogenase discovered in C. reinhardtii chloroplasts (Desplats et al., 2009). RNA interference lines expressing lower levels of NDA2 show lower hydrogen production rates, and it was concluded that NDA2 is involved in hydrogen production by the indirect pathway (Jans et al., 2008; Mignolet et al., 2012). The indirect pathway allows for an efficient time-based separation of O₂- and H₂-producing phases because it does not involve PSII activity and does not produce O₂. However, the indirect pathway has a much lower rate than the direct pathway (Cournac et al., 2002; Antal et al., 2009; Chochois et al., 2009). With the aim to identify limiting steps of hydrogen production in microalgae, we attempted to overexpress NDA2 in C. reinhardtii chloroplasts. We report that algal strains displaying a 2-fold increase in NDA2 show an increased nonphotochemical reduction of PQs and an increased rate of hydrogen production by the indirect pathway, the latter being only observed in conditions where stromal reducing equivalents are available in sufficient amounts.     

A Novel Method to Evaluate the Community Built Environment Using Photographs – Environmental Profile of a Community Health (EPOCH) Photo Neighbourhood Evaluation Tool

Background

Previous research has shown that environments with features that encourage walking are associated with increased physical activity. Existing methods to assess the built environment using geographical information systems (GIS) data, direct audit or large surveys of the residents face constraints, such as data availability and comparability, when used to study communities in countries in diverse parts of the world. The aim of this study was to develop a method to evaluate features of the built environment of communities using a standard set of photos. In this report we describe the method of photo collection, photo analysis instrument development and inter-rater reliability of the instrument.

Methods/Principal Findings

A minimum of 5 photos were taken per community in 86 communities in 5 countries according to a standard set of instructions from a designated central point of each community by researchers at each site. A standard pro forma derived from reviewing existing instruments to assess the built environment was developed and used to score the characteristics of each community. Photo sets from each community were assessed independently by three observers in the central research office according to the pro forma and the inter-rater reliability was compared by intra-class correlation (ICC). Overall 87% (53 of 60) items had an ICC of ≥0.70, 7% (4 of 60) had an ICC between 0.60 and 0.70 and 5% (3 of 60) items had an ICC ≤0.50.

Conclusions/Significance

Analysis of photos using a standardized protocol as described in this study offers a means to obtain reliable and reproducible information on the built environment in communities in very diverse locations around the world. The collection of the photographic data required minimal training and the analysis demonstrated high reliability for the majority of items of interest.     

Chromosomal initiation in Bacillus subtilis may involve two closely linked origins

Summary When the dnaB37 initiation mutant of Bacillus subtilis is returned to a permissive temperature following a period at 45° C, a synchronous round of DNA replication immediately ensues. Using this system we have been able to analyse the first fragments to be replicated while avoiding the use of thymine starvation or inhibitors of DNA replication. Such treatments are necessary to achieve even modest synchrony in germinating spores. Our results showed that the first fragment to be replicated was a 4kb BamHI-SalI restriction fragment, BS6. In contrast, when the analysis was performed out in the presence of novobiocin, an inhibitor of DNA gyrase, replication from BS6 was inhibited and the first fragment to be replicated was BS5, a 5.6 kb fragment located 1.7 kb to the right of BS 6. Replication from both putative origins was suppressed by rifamycin and was dependent upon dnaB. The results are discussed in relation to previous attempts to identify the first replicating fragment in germinating spores. We also discuss the possibility that B. subtilis contains two origins and suggest that either can act as the primary origin under certain conditions, or alternatively that both origins may act in concert in normal bidirectional replication, each site being required for the leading strand in each direction.     

Screening for Vulnerability in Older Cancer Patients: The ONCODAGE Prospective Multicenter Cohort Study

Background

Geriatric Assessment is an appropriate method for identifying older cancer patients at risk of life-threatening events during therapy. Yet, it is underused in practice, mainly because it is time- and resource-consuming. This study aims to identify the best screening tool to identify older cancer patients requiring geriatric assessment by comparing the performance of two short assessment tools the G8 and the Vulnerable Elders Survey (VES-13).

Patients and Methods

The diagnostic accuracy of the G8 and the (VES-13) were evaluated in a prospective cohort study of 1674 cancer patients accrued before treatment in 23 health care facilities. 1435 were eligible and evaluable. Outcome measures were multidimensional geriatric assessment (MGA), sensitivity (primary), specificity, negative and positive predictive values and likelihood ratios of the G8 and VES-13, and predictive factors of 1-year survival rate.

Results

Patient median age was 78.2 years (70-98) with a majority of females (69.8%), various types of cancer including 53.9% breast, and 75.8% Performance Status 0-1. Impaired MGA, G8, and VES-13 were 80.2%, 68.4%, and 60.2%, respectively. Mean time to complete G8 or VES-13 was about five minutes. Reproducibility of the two questionnaires was good. G8 appeared more sensitive (76.5% versus 68.7%, P =  0.0046) whereas VES-13 was more specific (74.3% versus 64.4%, P<0.0001). Abnormal G8 score (HR = 2.72), advanced stage (HR = 3.30), male sex (HR = 2.69) and poor Performance Status (HR = 3.28) were independent prognostic factors of 1-year survival.

Conclusion

With good sensitivity and independent prognostic value on 1-year survival, the G8 questionnaire is currently one of the best screening tools available to identify older cancer patients requiring geriatric assessment, and we believe it should be implemented broadly in daily practice. Continuous research efforts should be pursued to refine the selection process of older cancer patients before potentially life-threatening therapy.     

Recherches cytogénétiques sur les solanum,section tuberarium

Résumé Il ressort de divers essais de traitement de graines deS. Antipoviczii, par la colchicine, que la concentration optima est de l'ordre de 0,2%. La durée de l'immersion doit être d'environ 6 heures, s'il s'agit de graines prégermées; de 5 à 6 jours, s'il s'agit de graines sèches. Des observations analogues ont été faites, après traitements semblables, chezS. verrucosum etS. longipedicellatum.Le traitement, dans les conditions précitées, de graines deS. tuberosum, variétés Flava et Katahdin, a donné des plantes octoploïdes réduites, à feuilles coriaces, à pigmentation très foncée, terminalement ramifiées (Flava) ou non ramifiées (Katahdin); elles se sont montrées entièrement stériles pendant deux années consécutives, après avoir végété pendant 9 à 10 mois; leurs tubercules sont petits et peu nombreux.

---

Summary Various colchicine treatments of seeds, inS. Antipoviczii, have shown that the optimum concentration of the solution is 0,2%. Pre-germinated seeds should be soaked for about 6 hours and dry seeds 5 or 6 days, as is indicated through cytological analysis at various steps of the treatment. The same result have been obtained withS. verrucosum andS. longipedicellatum.After such a treatment, octoploids plants ofS. tuberosum var. Flava and var. Katahdin have been produced, which remain small and form thick dark-green leaves; the stem is thick, but very flexible, with dense terminal ramification (Flava) or without ramification (Katahdin). They produce a few small tubers but are completely sterible; in 1953 and in 1954, no flower has appeared even after a vegetative period of 9–10 months each year.

---

Samenvatting Een vergelijking van verschillende colchicine-concentraties, waarin zaden vanSolanum antipoviczii geweekt werden, heeft aangetoond dat de optimum-concentratie van de oplossing 0.2% bedraagt. Voorgekiemde zaden verlangen een onderdompeling van ongeveer zes uur, droge zaden vereisen vijf tot zes dagen, zoals bleek uit een cytologische analyse op verschillende tijdstippen tijdens de behandeling. Dezelfde resultaten werden verkregen metS. verrucosum en metS. longipedicellatum.Na zulk een behandeling werden octoploide planten vanS. tuberosum var. Flava en var. Katahdin verkregen, welke planten klein blijven en donkergroene, leerachtige bladeren vormen; de stengel is dik, maar goed buigbaar, met een dichte terminale vertakking (Flava) of onvertakt (Katahdin). Deze planten bleken twee jaar lang (1953 en 1954) volkomen steriel te zijn, in deze zin, dat ze na een vegetatieve groeitijd van 9–10 maanden niet tot bloei zijn gekomen. Wel produceerden ze talrijke kleine knolletjes.

---

---

Recherches subsidiées par le Fonds National de la Recherche Scientifique (Crédits aux Chercheurs).