Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

Design of new promoters and of a dual-bioreporter based on cross-activation by the two regulatory proteins XylR and HbpR.

The HbpR protein is the sigma54-dependent transcription activator for 2-hydroxybiphenyl degradation in Pseudomonas azelaica. The ability of HbpR and XylR, which share 35% amino acid sequence identity, to cross-activate the PhbpC and Pu promoters was investigated by determining HbpR- or XylR-mediated luciferase expression and by DNA binding assays. XylR measurably activated the PhbpC promoter in the presence of the effector m-xylene, both in Escherichia coli and Pseudomonas putida. HbpR weakly stimulated the Pu promoter in E. coli but not in P. azelaica. Poor HbpR-dependent activation from Pu was caused by a weak binding to the operator region. To create promoters efficiently activated by both regulators, the HbpR binding sites on PhbpC were gradually changed into the XylR binding sites of Pu by site-directed mutagenesis. Inducible luciferase expression from mutated promoters was tested in E. coli on a two plasmid system, and from mono copy gene fusions in P. azelaica and P. putida. Some mutants were efficiently activated by both HbpR and XylR, showing that promoters can be created which are permissive for both regulators. Others achieved a higher XylR-dependent transcription than from Pu itself. Mutants were also obtained which displayed a tenfold lower uninduced expression level by HbpR than the wild-type PhbpC, while keeping the same maximal induction level. On the basis of these results, a dual-responsive bioreporter strain of P. azelaica was created, containing both XylR and HbpR, and activating luciferase expression from the same single promoter independently with m-xylene and 2-hydroxybiphenyl.

EMDR-based treatment of psychotraumatic antecedents in illicit drug abusers: a report of two cases

The co-occurrence of PTSD and of substance use disorder (SD) is known to be very high. However the question of whether and how to treat such patients remains largely unanswered in the EMDR community. We report on two cases of EMDR-based treatment of heavily affected SD patients in whom psychotraumatic antecedents were identified. EMDR sessions focused on trauma-related material and not on the expression of cue-induced drug craving. The treatment appeared to be a difficult and challenging endeavour. However, some beneficial effects on general comfort and on drug consumption could be observed. A long stabilization phase was mandatory and the standard EMDR protocol needed to be conducted with much flexibility. Interestingly, there was no provocation of a prolonged psychological crisis or of relapse. Experiencing of emotional stress could be limited to the sessions and dissociation could be absorbed with specific well-known techniques without permanently increasing drug craving. These observations are discussed in relation to previously published concepts of using EMDR in the field of trauma and substance abuse.

Sensitive and rapid detection of ganciclovir resistance by PCR based MALDI-TOF analysis.

BACKGROUND: Cytomegalovirus (CMV) infection is associated with significant morbidity and mortality in transplant recipients. Resistance against ganciclovir is increasingly observed. According to current guidelines, direct drug resistance testing is not always performed due to high costs and work effort, even when resistance is suspected. OBJECTIVES: To develop a more sensitive, easy applicable and cost-effective assay as proof of concept for direct drug resistance testing in CMV surveillance of post-transplant patients. STUDY DESIGN: Five consecutive plasma samples from a heart transplant patient with a primary CMV infection were analyzed by quantitative real-time polymerase chain reaction (rtPCR) as a surrogate marker for therapy failure, and by direct drug resistance detection assays such as Sanger sequencing and the novel primer extension (PEX) reaction matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) based method. RESULTS: This report demonstrates that PEX reaction followed by MALDI-TOF analysis detects the A594V mutation, encoding ganciclovir resistance, ten days earlier compared to Sanger sequencing and more than 30 days prior to an increase in viral load. CONCLUSION: The greatly increased sensitivity and rapid turnaround-time combined with easy handling and moderate costs indicate that this procedure could make a major contribution to improve transplantation outcomes.

Expression of the NH(2)-terminal fragment of RasGAP in pancreatic beta-cells increases their resistance to stresses and protects mice from diabetes.

OBJECTIVE: Our laboratory has previously established in vitro that a caspase-generated RasGAP NH(2)-terminal moiety, called fragment N, potently protects cells, including insulinomas, from apoptotic stress. We aimed to determine whether fragment N can increase the resistance of pancreatic beta-cells in a physiological setting. RESEARCH DESIGN AND METHODS: A mouse line, called rat insulin promoter (RIP)-N, was generated that bears a transgene containing the rat insulin promoter followed by the cDNA-encoding fragment N. The histology, functionality, and resistance to stress of RIP-N islets were then assessed. RESULTS: Pancreatic beta-cells of RIP-N mice express fragment N, activate Akt, and block nuclear factor kappaB activity without affecting islet cell proliferation or the morphology and cellular composition of islets. Intraperitoneal glucose tolerance tests revealed that RIP-N mice control their glycemia similarly as wild-type mice throughout their lifespan. Moreover, islets isolated from RIP-N mice showed normal glucose-induced insulin secretory capacities. They, however, displayed increased resistance to apoptosis induced by a series of stresses including inflammatory cytokines, fatty acids, and hyperglycemia. RIP-N mice were also protected from multiple low-dose streptozotocin-induced diabetes, and this was associated with reduced in vivo beta-cell apoptosis. CONCLUSIONS: Fragment N efficiently increases the overall resistance of beta-cells to noxious stimuli without interfering with the physiological functions of the cells. Fragment N and the pathway it regulates represent, therefore, a potential target for the development of antidiabetes tools.

Example-based support vector machine for drug concentration analysis

Machine learning has been largely applied to analyze data in various domains, but it is still new to personalized medicine, especially dose individualization. In this paper, we focus on the prediction of drug concentrations using Support Vector Machines (S VM) and the analysis of the influence of each feature to the prediction results. Our study shows that SVM-based approaches achieve similar prediction results compared with pharmacokinetic model. The two proposed example-based SVM methods demonstrate that the individual features help to increase the accuracy in the predictions of drug concentration with a reduced library of training data.

Cost-effective experimental design to support modeling of concentration-response functions

Modeling concentration-response function became extremely popular in ecotoxicology during the last decade. Indeed, modeling allows determining the total response pattern of a given substance. However, reliable modeling is consuming in term of data, which is in contradiction with the current trend in ecotoxicology, which aims to reduce, for cost and ethical reasons, the number of data produced during an experiment. It is therefore crucial to determine experimental design in a cost-effective manner. In this paper, we propose to use the theory of locally D-optimal designs to determine the set of concentrations to be tested so that the parameters of the concentration-response function can be estimated with high precision. We illustrated this approach by determining the locally D-optimal designs to estimate the toxicity of the herbicide dinoseb on daphnids and algae. The results show that the number of concentrations to be tested is often equal to the number of parameters and often related to the their meaning, i.e. they are located close to the parameters. Furthermore, the results show that the locally D-optimal design often has the minimal number of support points and is not much sensitive to small changes in nominal values of the parameters. In order to reduce the experimental cost and the use of test organisms, especially in case of long-term studies, reliable nominal values may therefore be fixed based on prior knowledge and literature research instead of on preliminary experiments

Medication use in pregnancy: a cross-sectional, multinational web-based study.

OBJECTIVES: Intercountry comparability between studies on medication use in pregnancy is difficult due to dissimilarities in study design and methodology. This study aimed to examine patterns and factors associated with medications use in pregnancy from a multinational perspective, with emphasis on type of medication utilised and indication for use. DESIGN: Cross-sectional, web-based study performed within the period from 1 October 2011 to 29 February 2012. Uniform collection of drug utilisation data was performed via an anonymous online questionnaire. SETTING: Multinational study in Europe (Western, Northern and Eastern), North and South America and Australia. PARTICIPANTS: Pregnant women and new mothers with children less than 1 year of age. PRIMARY AND SECONDARY OUTCOME MEASURES: Prevalence of and factors associated with medication use for acute/short-term illnesses, chronic/long-term disorders and over-the-counter (OTC) medication use. RESULTS: The study population included 9459 women, of which 81.2% reported use of at least one medication (prescribed or OTC) during pregnancy. Overall, OTC medication use occurred in 66.9% of the pregnancies, whereas 68.4% and 17% of women reported use of at least one medication for treatment of acute/short-term illnesses and chronic/long-term disorders, respectively. The extent of self-reported medicated illnesses and types of medication used by indication varied across regions, especially in relation to urinary tract infections, depression or OTC nasal sprays. Women with higher age or lower educational level, housewives or women with an unplanned pregnancy were those most often reporting use of medication for chronic/long-term disorders. Immigrant women in Western (adjusted OR (aOR): 0.55, 95% CI 0.34 to 0.87) and Northern Europe (aOR: 0.50, 95% CI 0.31 to 0.83) were less likely to report use of medication for chronic/long-term disorders during pregnancy than non-immigrants. CONCLUSIONS: In this study, the majority of women in Europe, North America, South America and Australia used at least one medication during pregnancy. There was a substantial inter-region variability in the types of medication used.

General unknown screening procedure for the characterization of human drug metabolites: application to loratadine phase I metabolism.

This article describes the application of a recently developed general unknown screening (GUS) strategy based on LC coupled to a hybrid linear IT-triple quadrupole mass spectrometer (LC-MS/MS-LIT) for the simultaneous detection and identification of drug metabolites following in vitro incubation with human liver microsomes. The histamine H1 receptor antagonist loratadine was chosen as a model compound to demonstrate the interest of such approach, because of its previously described complex and extensive metabolism. Detection and mass spectral characterization were based on data-dependent acquisition, switching between a survey scan acquired in the ion-trapping Q3 scan mode with dynamic subtraction of background noise, and a dependent scan in the ion-trapping product ion scan mode of automatically selected parent ions. In addition, the MS(3) mode was used in a second step to confirm the structure of a few fragment ions. The sensitivity of the ion-trapping modes combined with the selectivity of the triple quadrupole modes allowed, with only one injection, the detection and identification of 17 phase I metabolites of loratadine. The GUS procedure used in this study may be applicable as a generic technique for the characterization of drug metabolites after in vitro incubation, as well as probably in vivo experiments.

Test result-based sampling: an efficient design for estimating the accuracy of patient safety indicators.

OBJECTIVE: Accuracy studies of Patient Safety Indicators (PSIs) are critical but limited by the large samples required due to low occurrence of most events. We tested a sampling design based on test results (verification-biased sampling [VBS]) that minimizes the number of subjects to be verified. METHODS: We considered 3 real PSIs, whose rates were calculated using 3 years of discharge data from a university hospital and a hypothetical screen of very rare events. Sample size estimates, based on the expected sensitivity and precision, were compared across 4 study designs: random and VBS, with and without constraints on the size of the population to be screened. RESULTS: Over sensitivities ranging from 0.3 to 0.7 and PSI prevalence levels ranging from 0.02 to 0.2, the optimal VBS strategy makes it possible to reduce sample size by up to 60% in comparison with simple random sampling. For PSI prevalence levels below 1%, the minimal sample size required was still over 5000. CONCLUSIONS: Verification-biased sampling permits substantial savings in the required sample size for PSI validation studies. However, sample sizes still need to be very large for many of the rarer PSIs.

Randomized comparison of biodegradable polymer sirolimus-eluting stents versus durable polymer everolimus-eluting stents for percutaneous coronary revascularization: Rationale and design of the BIOSCIENCE trial.

BACKGROUND: Biodegradable polymers for release of antiproliferative drugs from metallic drug-eluting stents aim to improve long-term vascular healing and efficacy. We designed a large scale clinical trial to compare a novel thin strut, cobalt-chromium drug-eluting stent with silicon carbide-coating releasing sirolimus from a biodegradable polymer (O-SES, Orsiro; Biotronik, Bülach, Switzerland) with the durable polymer-based Xience Prime/Xpedition everolimus-eluting stent (EES) (Xience Prime/Xpedition stent, Abbott Vascular, IL) in an all-comers patient population. DESIGN: The multicenter BIOSCIENCE trial (NCT01443104) randomly assigned 2,119 patients to treatment with biodegradable polymer sirolimus-eluting stents (SES) or durable polymer EES at 9 sites in Switzerland. Patients with chronic stable coronary artery disease or acute coronary syndromes, including non-ST-elevation and ST-elevation myocardial infarction, were eligible for the trial if they had at least 1 lesion with a diameter stenosis >50% appropriate for coronary stent implantation. The primary end point target lesion failure (TLF) is a composite of cardiac death, target vessel myocardial infarction, and clinically driven target lesion revascularization within 12 months. Assuming a TLF rate of 8% at 12 months in both treatment arms and accepting 3.5% as a margin for noninferiority, inclusion of 2,060 patients would provide more than 80% power to detect noninferiority of the biodegradable polymer SES compared with the durable polymer EES at a 1-sided type I error of 0.05. Clinical follow-up will be continued through 5 years. CONCLUSION: The BIOSCIENCE trial will determine whether the biodegradable polymer SES is noninferior to the durable polymer EES with respect to TLF.

Design of new tools to improve the efficacy of DNA-damaging drugs used in cancer therapy

Résumé Les tumeurs sont diverses et hétérogènes, mais toutes partagent la capacité de proliférer sans contrôle. Une prolifération dérégulée de cellules couplée à une insensibilité à une réponse apoptotique constitue une condition minimale pour que l'évolution d'une tumeur se produise. Un des traitements les plus utilisés pour traité le cancer à l'heure actuelle sont les chimiothérapies, qui sont fréquemment des composés chimiques qui induisent des dommages dans l'ADN. Les agents anticancéreux sont efficaces seulement quand les cellules tumorales sont plus aisément tuées que le tissu normal environnant. L'efficacité de ces agents est en partie déterminée par leur capacité à induire l'apoptose. Nous avons récemment démontré que la protéine RasGAP est un substrat non conventionnel des caspases parce elle peut induire à la fois des signaux anti et pro-apoptotiques, selon l'ampleur de son clivage par les caspases. A un faible niveau d'activité des caspases, RasGAP est clivé, générant deux fragments (le fragment N et le fragment C). Le fragment N semble être un inhibiteur général de l'apoptose en aval de l'activation des caspases. À des niveaux plus élevés d'activité des caspases, la capacité du fragment N de contrecarrer l'apoptose est supprimée quand il est clivé à nouveau par les caspases. Ce dernier clivage produit deux nouveaux fragments, N 1 et N2, qui contrairement au fragment N sensibilisent efficacement des cellules cancéreuses envers des agents chimiothérapeutiques. Dans cette étude nous avons prouvé qu'un peptide, appelé par la suite TAT-RasGAP317-326, qui est dérivé du fragment N2 de RasGAP et est rendu perméable aux cellules, sensibilise spécifiquement des cellules cancéreuses à trois génotoxines différentes utilisées couramment dans des traitements anticancéreux, et cela dans des modèles in vitro et in vivo. Il est important de noté que ce peptide semble ne pas avoir d'effet sur des cellules non cancéreuses. Nous avons également commencé à caractériser les mécanismes moléculaires expliquant les fonctions de sensibilisation de TAT-RasGAP317-326. Nous avons démontré que le facteur de transcription p53 et une protéine sous son activité transcriptionelle, nommée Puma, sont indispensables pour l'activité de TAT-RasGAP317-326. Nous avons également prouvé que TAT-RasGAP317-326 exige la présence d'une protéine appelée G3BP1, une protéine se liant a RasGAP, pour potentialisé les effets d'agents anticancéreux. Les données obtenues dans cette étude montrent qu'il pourrait être possible d'augmenter l'efficacité des chimiothérapies à l'aide d'un composé capable d'augmenter la sensibilité des tumeurs aux génotoxines et ainsi pourrait permettre de traiter de manière plus efficace des patients sous traitement chimiothérapeutiques. Summary Tumors are diverse and heterogeneous, but all share the ability to proliferate without control. Deregulated cell proliferation coupled with suppressed apoptotic sensitivity constitutes a minimal requirement upon which tumor evolution occurs. One of the most commonly used treatments is chemotherapy, which frequently uses chemical compounds that induce DNA damages. Anticancer agents are effective only when tumors cells are more readily killed than the surrounding normal tissue. The efficacy of these agents is partly determined by their ability to induce apoptosis. We have recently demonstrated that the protein RasGAP is an unconventional caspase substrate because it can induce both anti- and pro-apoptotic signals, depending on the extent of its cleavage by caspases. At low levels of caspase activity, RasGAP is cleaved, generating an N-terminal fragment (fragment N) and a C-terminal fragment (fragment C). Fragment N appears to be a general Mocker of apoptosis downstream of caspase activation. At higher levels of caspase activity, the ability of fragment N to counteract apoptosis is suppressed when it is further cleaved. This latter cleavage event generates two fragments, N1 and N2, which in contrast to fragment N potently sensitizes cancer cells toward DNA-damaging agents induced apoptosis. In the present study we show that a cell permeable peptide derived from the N2 fragment of RasGAP, thereafter called TAT-RasGAP317-326, specifically sensitizes cancer cells to three different genotoxins commonly used in chemotherapy in vitro and in vivo models. Importantly this peptide seems not to have any effect on non cancer cells. We have also started to characterize the molecular mechanisms underlying the sensitizing functions of TAT-RasGAP317-326. We have demonstrated that the p53 transcription factor and a protein under its transcriptional activity, called Puma, are required for the activity of TATRasGAP317-326. We have also showed that TAT-RasGAP317-326 requires the presence of a protein called G3BP1, which have been shown to interact with RasGAP, to increase the effect of the DNA-damaging drug cisplatin. The data obtained in this study showed that it is possible to increase the efficacy of current used chemotherapies with a compound able to increase the efficacy of genotoxins which could be beneficial for patients subjected to chemotherapy.

Business model design: an evaluation of paper-based and computer-aided canvases

In recent years, Business Model Canvas design has evolved from being a paper-based activity to one that involves the use of dedicated computer-aided business model design tools. We propose a set of guidelines to help design more coherent business models. When combined with functionalities offered by CAD tools, they show great potential to improve business model design as an ongoing activity. However, in order to create complex solutions, it is necessary to compare basic business model design tasks, using a CAD system over its paper-based counterpart. To this end, we carried out an experiment to measure user perceptions of both solutions. Performance was evaluated by applying our guidelines to both solutions and then carrying out a comparison of business model designs. Although CAD did not outperform paper-based design, the results are very encouraging for the future of computer-aided business model design.

Evaluation of drug-induced neurotoxicity based on metabolomics, proteomics and electrical activity measurements in complementary CNS in vitro models.

The present study was performed in an attempt to develop an in vitro integrated testing strategy (ITS) to evaluate drug-induced neurotoxicity. A number of endpoints were analyzed using two complementary brain cell culture models and an in vitro blood-brain barrier (BBB) model after single and repeated exposure treatments with selected drugs that covered the major biological, pharmacological and neuro-toxicological responses. Furthermore, four drugs (diazepam, cyclosporine A, chlorpromazine and amiodarone) were tested more in depth as representatives of different classes of neurotoxicants, inducing toxicity through different pathways of toxicity. The developed in vitro BBB model allowed detection of toxic effects at the level of BBB and evaluation of drug transport through the barrier for predicting free brain concentrations of the studied drugs. The measurement of neuronal electrical activity was found to be a sensitive tool to predict the neuroactivity and neurotoxicity of drugs after acute exposure. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found to be well suited for OMICs analyses after both acute and long term treatment. The obtained data suggest that an in vitro ITS based on the information obtained from BBB studies and combined with metabolomics, proteomics and neuronal electrical activity measurements performed in stable in vitro neuronal cell culture systems, has high potential to improve current in vitro drug-induced neurotoxicity evaluation.