Genome-wide screen of genes required for caffeine tolerance in fission yeast

Background: An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to atoxic dose of this drug, and the relationship between caffeine and oxidative stress pathways.Methodology/Principal Findings: We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeinecontainingplates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H2O2-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 aresensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner. Conclusions/Significance: With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we havedemonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.

Sustained release of nanosized complexes of polyethylenimine and anti-TGF-beta 2 oligonucleotide improves the outcome of glaucoma surgery.

The purpose of this study was to design microspheres combining sustained delivery and enhanced intracellular penetration for ocular administration of antisense oligonucleotides. Nanosized complexes of antisense TGF-beta2 phosphorothioate oligonucleotides (PS-ODN) with polyethylenimine (PEI), and naked PS-ODN were encapsulated into poly(lactide-co-glycolide) microspheres prepared by the double-emulsion solvent evaporation method. The PS-ODN was introduced either naked or complexed in the inner aqueous phase of the first emulsion. We observed a marked influence of microsphere composition on porosity, size distribution and PS-ODN encapsulation efficiency. Mainly, the presence of PEI induced the formation of large pores observed onto microsphere surface. Introduction of NaCl in the outer aqueous phase increased the encapsulation efficiency and reduced microsphere porosity. In vitro release kinetic of PS-ODN was also investigated. Clearly, the higher the porosity, the faster was the release and the higher was the burst effect. Using an analytical solution of Fick's second law of diffusion, it was shown that the early phase of PS-ODN and PS-ODN-PEI complex release was primarily controlled by pure diffusion, irrespectively of the type of microsphere. Finally, microspheres containing antisense TGF-beta2 nanosized complexes were shown, after subconjunctival administration to rabbit, to significantly increase intracellular penetration of ODN in conjunctival cells and subsequently to improve bleb survival in a rabbit experimental model of filtering surgery. These results open up interesting prospective for the local controlled delivery of genetic material into the eye.

Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors.

We describe a new mechanism regulating the tumor endothelial barrier and T cell infiltration into tumors. We detected selective expression of the death mediator Fas ligand (FasL, also called CD95L) in the vasculature of human and mouse solid tumors but not in normal vasculature. In these tumors, FasL expression was associated with scarce CD8(+) infiltration and a predominance of FoxP3(+) T regulatory (Treg) cells. Tumor-derived vascular endothelial growth factor A (VEGF-A), interleukin 10 (IL-10) and prostaglandin E2 (PGE2) cooperatively induced FasL expression in endothelial cells, which acquired the ability to kill effector CD8(+) T cells but not Treg cells because of higher levels of c-FLIP expression in Treg cells. In mice, genetic or pharmacologic suppression of FasL produced a substantial increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells. Pharmacologic inhibition of VEGF and PGE2 produced a marked increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells that was dependent on attenuation of FasL expression and led to CD8-dependent tumor growth suppression. Thus, tumor paracrine mechanisms establish a tumor endothelial death barrier, which has a critical role in establishing immune tolerance and determining the fate of tumors.

Pivotal role for a tail subunit of the RNA polymerase II mediator complex CgMed2 in azole tolerance and adherence in Candida glabrata.

Antifungal therapy failure can be associated with increased resistance to the employed antifungal agents. Candida glabrata, the second most common cause of invasive candidiasis, is intrinsically less susceptible to the azole class of antifungals and accounts for 15% of all Candida bloodstream infections. Here, we show that C. glabrata MED2 (CgMED2), which codes for a tail subunit of the RNA polymerase II Mediator complex, is required for resistance to azole antifungal drugs in C. glabrata. An inability to transcriptionally activate genes encoding a zinc finger transcriptional factor, CgPdr1, and multidrug efflux pump, CgCdr1, primarily contributes to the elevated susceptibility of the Cgmed2Δ mutant toward azole antifungals. We also report for the first time that the Cgmed2Δ mutant exhibits sensitivity to caspofungin, a constitutively activated protein kinase C-mediated cell wall integrity pathway, and elevated adherence to epithelial cells. The increased adherence of the Cgmed2Δ mutant was attributed to the elevated expression of the EPA1 and EPA7 genes. Further, our data demonstrate that CgMED2 is required for intracellular proliferation in human macrophages and modulates survival in a murine model of disseminated candidiasis. Lastly, we show an essential requirement for CgMed2, along with the Mediator middle subunit CgNut1 and the Mediator cyclin-dependent kinase/cyclin subunit CgSrb8, for the high-level fluconazole resistance conferred by the hyperactive allele of CgPdr1. Together, our findings underscore a pivotal role for CgMed2 in basal tolerance and acquired resistance to azole antifungals.

Importance of genotypic and phenotypic tolerance in the treatment of experimental endocarditis due to Streptococcus gordonii.

Genotypic and phenotypic tolerance was studied in penicillin treatment of experimental endocarditis due to nontolerant and tolerant Streptococcus gordonii and to their backcross transformants. The organisms were matched for in vitro and in vivo growth rates. Rats with aortic endocarditis were treated for 3 or 5 days, starting 12, 24, or 48 h after inoculation. When started at 12 h, during fast intravegetation growth, 3 days of treatment cured 80% of the nontolerant parent compared with <30% of the tolerant derivative (P < .005). When started at 24 or 48 h and if intravegetation growth had reached a plateau, 3 days of treatment failed against both bacteria. However, a significant difference between the 2 organisms was restored when treatment was extended to 5 days. Thus, genotypic tolerance conferred a survival advantage in both fast- and slow-growing bacteria, demonstrating that the in vitro-defined tolerant phenotype also carried the risk of treatment failure in vivo.

Error and attack tolerance of layered complex networks.

Many complex systems may be described by not one but a number of complex networks mapped on each other in a multi-layer structure. Because of the interactions and dependencies between these layers, the state of a single layer does not necessarily reflect well the state of the entire system. In this paper we study the robustness of five examples of two-layer complex systems: three real-life data sets in the fields of communication (the Internet), transportation (the European railway system), and biology (the human brain), and two models based on random graphs. In order to cover the whole range of features specific to these systems, we focus on two extreme policies of system's response to failures, no rerouting and full rerouting. Our main finding is that multi-layer systems are much more vulnerable to errors and intentional attacks than they appear from a single layer perspective.

Active intravenous drug use during chronic hepatitis C therapy does not reduce sustained virological response rates in adherent patients.

SUMMARY: Reluctance has been expressed about treating chronic hepatitis C in active intravenous (IV) drug users (IDUs), and this is found in both international guidelines and routine clinical practice. However, the medical literature provides no evidence for an unequivocal treatment deferral of this risk group. We retrospectively analyzed the direct effect of IV drug use on treatment outcome in 500 chronic hepatitis C patients enrolled in the Swiss Hepatitis C Cohort Study. Patients were eligible for the study if they had their serum hepatitis C virus (HCV) RNA tested 6 months after the end of treatment and at least one visit during the antiviral therapy, documenting the drug use status. Five hundred patients fulfilled the inclusion criteria (199 were IDU and 301 controls). A minimum exposure to 80% of the scheduled cumulative dose of antivirals was reached in 66.0% of IDU and 60.5% of controls (P = NS). The overall sustained virological response (SVR) rate was 63.6%. Active IDU reached a SVR of 69.3%, statistically not significantly different from controls (59.8%). A multivariate analysis for treatment success showed no significant negative influence of active IV drug use. In conclusion, our study shows no relevant direct influence of IV drugs on the efficacy of anti-HCV therapy among adherent patients.

Dexamethasone-loaded poly(epsilon-caprolactone) intravitreal implants: a pilot study.

PURPOSE: Poly(epsilon-caprolactone) (PCL) is a biodegradable and biocompatible polymer that presents a very low degradation rate, making it suitable for the development of long-term drug delivery systems. The objective of this pilot study is to evaluate the feasibility and characteristics of PCL devices in the prolonged and controlled intravitreous release of dexamethasone. METHODS: The in vitro release of dexamethasone was investigated and the implant degradation was monitored by the percent of mass loss and by changes in the surface morphology. Differential scanning calorimetry was used to evaluate stability and interaction of the implant and the drug. The short-term tolerance of the implants was studied after intravitreous implantation in rabbit eye. Results: PCL implant allows for a controlled and prolonged delivery of dexamethasone since it releases 25% of the drug in 21 weeks. Its low degradation rate was confirmed by the mass loss and scanning electron microscopy studies. Preliminary observations show that PCL intravitreous implants are very well tolerated in the rabbit eye. CONCLUSION: This study demonstrates the PCL drug delivery systems allowed to a prolonged release of dexamethasone in vitro. The implants demonstrated a strikingly good intraocular short-term tolerance in rabbits eyes. The in vitro and preliminary in vivo studies tend to show that PCL implants could be of interest when long-term sustained intraocular delivery of corticosteroids is required.

The sigma factor AlgU (AlgT) controls exopolysaccharide production and tolerance towards desiccation and osmotic stress in the biocontrol agent Pseudomonas fluorescens CHA0.

A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases. This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and sigma(22)) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent Pseudomonas fluorescens CHA0. The algU-mucA-mucB gene cluster of strain CHA0 was similar to that of the pathogens Pseudomonas aeruginosa and Pseudomonas syringae. Strain CHA0 is naturally nonmucoid, whereas a mucA deletion mutant or algU-overexpressing strains were highly mucoid due to exopolysaccharide overproduction. Mucoidy strictly depended on the global regulator GacA. An algU deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the mucA mutant were. Expression of an algU'-'lacZ reporter fusion was induced severalfold in the wild type and in the mucA mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in the algU mutant. Overexpression of algU did not enhance tolerance towards osmotic stress. AlgU was found to be essential for tolerance of P. fluorescens towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil. The size of the population of the algU mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5%. In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of P. fluorescens towards oxidative and heat stress. In conclusion, AlgU is a crucial determinant in the adaptation of P. fluorescens to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment.

Mononuclear cell transcriptome response after sustained virgin olive oil consumption in humans: an exploratory nutrigenomics study

Virgin olive oil (VOO) is considered to be one of the main components responsible for the health benefits of the Mediterranean diet, particularly against atherosclerosis where peripheral blood mononuclear cells (PBMNCs) play a crucial role in atherosclerosis development and progression. The objective of this article was to identify the PBMNC genes that respond to VOO consumption in order to ascertain the molecular mechanisms underlying the beneficial action of VOO in the prevention of atherosclerosis. Gene expression profiles of PBMNCs from healthy individuals were examined in pooled RNA samples by microarrays after 3 weeks of moderate and regular consumption of VOO, as the main fat source in a diet controlled for antioxidant content. Gene expression was verified by qPCR. The response to VOO consumption was confirmed for individual samples (n = 10) by qPCR for 10 upregulated genes (ADAM17, ALDH1A1, BIRC1, ERCC5, LIAS, OGT, PPARBP, TNFSF10, USP48, and XRCC5). Their putative role in the molecular mechanisms involved in atherosclerosis development and progression is discussed, focusing on a possible relation with VOO consumption. Our data support the hypothesis that 3 weeks of nutritional intervention with VOO supplementation, at doses common in the Mediterranean diet, can alter the expression of genes related to atherosclerosis development and progression.

Tolerance of whitefish embryos to Pseudomonas fluorescens linked to genetic and maternal effects, and reduced by previous exposure.

Juvenile or adult fish can alter their behaviour and rely on an innate and adaptive immune system to avoid/counteract pathogens, while fish embryos have to depend on egg characteristics and may be partly protected by their developing immune system that is building up from a certain age on. We developed an infection protocol that allows testing the reaction of individual whitefish embryos (Coregonus palaea) to repeated exposures to Pseudomonas fluorescens, an opportunistic bacterial fish pathogen. We used a full-factorial in vitro breeding design to separately test the effects of paternal and maternal contributions to the embryos' susceptibility to different kinds of pathogen exposure. We found that a first non-lethal exposure had immunosuppressive effects: pre-exposed embryos were more susceptible to future challenges with the same pathogen. At intermediate and high levels of pathogen intensity, maternal effects turned out to be crucial for the embryos' tolerance to infection. Paternal (i.e. genetic) effects played a significant role at the strongest level of infection, i.e. the embryos' own genetics already explained some of the variation in embryo susceptibility. Our findings suggest that whitefish embryos are largely protected by maternally transmitted substances, but build up some own innate immunocompetence several days before hatching.

Dose-dependent acute and sustained renal effects of the endothelin receptor antagonist avosentan in healthy subjects

Enjeu : L'incidence d'insuffisance rénale terminale augmente d'environ 5-6% par année dans nos régions. L'une des causes majeures d'insuffisance rénale est la néphropathie diabétique qui représente selon les pays entre 25 et 40% des néphropathies terminales. La progression de la néphropathie diabétique peut être ralentie de manière efficace par un bon contrôle du diabète et de l'hypertension artérielle et par le blocage du système rénine-angiotensine. Néanmoins, malgré l'application stricte de ces thérapies préventives, la néphropathie de bons nombres de patients diabétiques continue de progresser. Il est donc important de développer de nouvelles stratégies permettant de préserver la fonction rénale des patients diabétiques soit en améliorant le contrôle de la pression artérielle soit en diminuant la protéinurie. Contexte : Il existe un certain nombre d'évidences expérimentales que le blocage des récepteurs de l'endothéline pourrait avoir un effet positif sur le devenir de la néphropathie diabétique en diminuant de manière efficace la protéinurie même chez des animaux déjà traités efficacement avec un bloqueur du système rénine-angiotensine. Dans des études de phase 2 impliquant l'avosentan, un antagoniste des récepteurs de l'endothéline actuellement en cours de développement pour le traitement de la néphropathie diabétique, on a pu démontrer que cet antagoniste, prescrit à des doses oscillant entre 5 et 50 mg par jour per os, diminue la protéinurie d'environ 20-40% chez des patients déjà traités avec un IEC ou un antagoniste de l'angiotensine. Toutefois, une grande étude de phase III conduite avec ce médicament chez des patients diabétiques a du être interrompue précocement en raison de l'apparition d'oedèmes et d'une surcharge hydrosodée conduisant dans certains cas à une décompensation cardiaque aiguë. La rétention hydrosodée est un effet secondaire connu des antagonistes de l'endothéline déjà sur le marché. Toutefois, pour l'avosentan, on ne savait pas si des doses plus faibles du médicament avaient aussi un effet négative sur la balance hydrosodée. En outre, les mécanismes rénaux responsables de la rétention hydrosodée sont encore mal connus chez l'homme. C'est pourquoi, nous avons organisé et réalisé cette étude de pharmacologie clinique chez le volontaire sain posant 2 questions : 1) des doses faibles d'avosentan produisent-elles aussi une rétention hydrosodée chez l'homme ? et 2) quels sont les mécanismes rénaux pouvant expliquer la rétention hydrosodée ? Cette thèse est donc une étude clinique de phase I testant chez 23 volontaires sains les effets rénaux de différentes doses d'avosentan ou d'un placebo pour établir la courbe dose-réponse des effets rénaux de ce médicament. L'idée était également de définir quelle dose est sure et bien tolérée pour être utilisée dans une nouvelle étude de phase II. L'avosentan a été administré par voie orale une fois par jour pendant 8 jours à des doses de 0.5, 1.5, 5 et 50 mg. Les effets rénaux hémodynamiques et tubulaires ont été étudiés chez chaque sujet lors de la première administration (jour 1) et après une semaine de traitement (jour 8). Le médicament a induit une prise de poids dose-dépendante déjà présente à 5 mg et maximale à 50 mg (+ 0.8 kg au jour 8). Nous n'avons pas mesuré d'impact de l'avosentan sur l'hémodynamique rénale ni sur les électrolytes plasmatiques. En revanche, nous avons constaté une diminution dose-dépendante de la fraction d'excrétion de sodium (jusqu'à -8.7% avec avosentan 50 mg). Cette diminution était en rapport avec une augmentation dose-dépendante de la réabsorption proximale de sodium. Nous avons également constaté une baisse de la pression artérielle aux doses élevées et une hémodilution marquée par une baisse de l'hématocrite suggérant une rétention hydrique à la plus haute dose. Nos résultats suggèrent donc que l'avosentan induit une rétention sodée rénale dose-dépendante expliquée avant tout par une rétention du sodium au niveau du tubule proximal. Cet effet n'est pas observé à des doses plus basses que 5 mg chez le volontaire sain, suggérant que ce médicament devrait être évalué pour son activité réno-protectrice à des doses inférieures ou égales à 5 mg par jour. La raison pour laquelle les hautes doses produisent plus de rétention sodée est peut être liée à une perte de sélectivité pour les sous-types (A et B) de récepteurs à l'endothéline lorsque l'on administre des doses plus élevées que 5 mg. Perspectives : Les résultats de ce travail de thèse ont donc permis de caractériser les propriétés rénales d'un nouvel antagoniste des récepteurs de l'endothéline chez l'homme. Ces résultats ont aussi permis de guider le développement futur de ce médicament vers des doses plus faibles avec l'espoir de garder les effets bénéfiques sur la protéinurie tout en améliorant le profil de tolérance du médicament par l'utilisation de doses plus faibles. ANGLAIS The endothelin receptor antagonist avosentan may cause fluid overload at doses of 25 and 50 mg, but the actual mechanisms of this effect are unclear. We conducted a placebo-controlled study in 23 healthy subjects to assess the renal effects of avosentan and the dose dependency of these effects. Oral avosentan was administered once daily for 8 days at doses of 0.5, 1.5, 5, and 50 mg. The drug induced a dose-dependent median increase in body weight, most pronounced at 50 mg (0.8 kg on day 8). Avosentan did not affect renal hemodynamics or plasma electrolytes. A dose-dependent median reduction in the fractional renal excretion of sodium was found (up to 8.7% at avosentan 50 mg); this reduction was paralleled by a dose-related increase in proximal sodium reabsorption. It is suggested that avosentan dose-dependently induces sodium retention by the kidney, mainly through proximal tubular effects. The potential clinical benefits of avosentan should therefore be investigated at doses of ≤ 5 mg.

Disentangling human tolerance and resistance against HIV.

In ecology, "disease tolerance" is defined as an evolutionary strategy of hosts against pathogens, characterized by reduced or absent pathogenesis despite high pathogen load. To our knowledge, tolerance has to date not been quantified and disentangled from host resistance to disease in any clinically relevant human infection. Using data from the Swiss HIV Cohort Study, we investigated if there is variation in tolerance to HIV in humans and if this variation is associated with polymorphisms in the human genome. In particular, we tested for associations between tolerance and alleles of the Human Leukocyte Antigen (HLA) genes, the CC chemokine receptor 5 (CCR5), the age at which individuals were infected, and their sex. We found that HLA-B alleles associated with better HIV control do not confer tolerance. The slower disease progression associated with these alleles can be fully attributed to the extent of viral load reduction in carriers. However, we observed that tolerance significantly varies across HLA-B genotypes with a relative standard deviation of 34%. Furthermore, we found that HLA-B homozygotes are less tolerant than heterozygotes. Lastly, tolerance was observed to decrease with age, resulting in a 1.7-fold difference in disease progression between 20 and 60-y-old individuals with the same viral load. Thus, disease tolerance is a feature of infection with HIV, and the identification of the mechanisms involved may pave the way to a better understanding of pathogenesis.

One-sided tolerance interval in a two-way balanced nested model with mixed effects

In many research areas (such as public health, environmental contamination, and others) one deals with the necessity of using data to infer whether some proportion (%) of a population of interest is (or one wants it to be) below and/or over some threshold, through the computation of tolerance interval. The idea is, once a threshold is given, one computes the tolerance interval or limit (which might be one or two - sided bounded) and then to check if it satisfies the given threshold. Since in this work we deal with the computation of one - sided tolerance interval, for the two-sided case we recomend, for instance, Krishnamoorthy and Mathew [5]. Krishnamoorthy and Mathew [4] performed the computation of upper tolerance limit in balanced and unbalanced one-way random effects models, whereas Fonseca et al [3] performed it based in a similar ideas but in a tow-way nested mixed or random effects model. In case of random effects model, Fonseca et al [3] performed the computation of such interval only for the balanced data, whereas in the mixed effects case they dit it only for the unbalanced data. For the computation of twosided tolerance interval in models with mixed and/or random effects we recomend, for instance, Sharma and Mathew [7]. The purpose of this paper is the computation of upper and lower tolerance interval in a two-way nested mixed effects models in balanced data. For the case of unbalanced data, as mentioned above, Fonseca et al [3] have already computed upper tolerance interval. Hence, using the notions persented in Fonseca et al [3] and Krishnamoorthy and Mathew [4], we present some results on the construction of one-sided tolerance interval for the balanced case. Thus, in order to do so at first instance we perform the construction for the upper case, and then the construction for the lower case.

Tolerance and reactivity of NK cells: two sides of the same coin?

NK cells can kill transformed, infected and stressed cells while most normal cells are spared. NK cells are activated by various endogenous self-ligands, some of which are actually expressed by normal cells. Thus, NK cells are inherently self-reactive and consequently, potentially auto-aggressive. How these cells are prevented from attacking normal cells while ensuring reactivity to diseased cells is a major unresolved question for NK-cell biologists.