PEG-b-PPS diblock copolymer aggregates for hydrophobic drug solubilization and release: cyclosporin A as an example

Micelles formed from amphiphilic block copolymers have been explored in recent years as carriers for hydrophobic drugs. In an aqueous environment, the hydrophobic blocks form the core of the micelle, which can host lipophilic drugs, while the hydrophilic blocks form the corona or outer shell and stabilize the interface between the hydrophobic core and the external medium. In the present work, mesophase behavior and drug encapsulation were explored in the AB block copolymeric amphiphile composed of poly(ethylene glycol) (PEG) as a hydrophile and poly(propylene sulfide) PPS as a hydrophobe, using the immunosuppressive drug cyclosporin A (CsA) as an example of a highly hydrophobic drug. Block copolymers with a degree of polymerization of 44 on the PEG and of 10, 20 and 40 on the PPS respectively (abbreviated as PEG44-b-PPS10, PEG44-b-PPS20, PEG44-b-PPS40) were synthesized and characterized. Drug-loaded polymeric micelles were obtained by the cosolvent displacement method as well as the remarkably simple method of dispersing the warm polymer melt, with drug dissolved therein, in warm water. Effective drug solubility up to 2 mg/mL in aqueous media was facilitated by the PEG- b-PPS micelles, with loading levels up to 19% w/w being achieved. Release was burst-free and sustained over periods of 9-12 days. These micelles demonstrate interesting solubilization characteristics, due to the low glass transition temperature, highly hydrophobic nature, and good solvent properties of the PPS block

An ancient founder mutation in PROKR2 impairs human reproduction.

Congenital gonadotropin-releasing hormone (GnRH) deficiency manifests as absent or incomplete sexual maturation and infertility. Although the disease exhibits marked locus and allelic heterogeneity, with the causal mutations being both rare and private, one causal mutation in the prokineticin receptor, PROKR2 L173R, appears unusually prevalent among GnRH-deficient patients of diverse geographic and ethnic origins. To track the genetic ancestry of PROKR2 L173R, haplotype mapping was performed in 22 unrelated patients with GnRH deficiency carrying L173R and their 30 first-degree relatives. The mutation's age was estimated using a haplotype-decay model. Thirteen subjects were informative and in all of them the mutation was present on the same ~123 kb haplotype whose population frequency is ≤10%. Thus, PROKR2 L173R represents a founder mutation whose age is estimated at approximately 9000 years. Inheritance of PROKR2 L173R-associated GnRH deficiency was complex with highly variable penetrance among carriers, influenced by additional mutations in the other PROKR2 allele (recessive inheritance) or another gene (digenicity). The paradoxical identification of an ancient founder mutation that impairs reproduction has intriguing implications for the inheritance mechanisms of PROKR2 L173R-associated GnRH deficiency and for the relevant processes of evolutionary selection, including potential selective advantages of mutation carriers in genes affecting reproduction.

Primetime for antiangiogenic therapy.

PURPOSE OF REVIEW: To review the current experience with angiogenesis inhibitors in the treatment of gliomas. RECENT FINDINGS: Antiangiogenic therapy has recently reached the clinic with the approval of bevacizumab for recurrent glioblastomas. A number of promising antiangiogenic and vasculature-modifying agents are under investigation for newly diagnosed and recurrent malignant gliomas. A recurrence under ongoing or after antiangiogenic therapy is often characterized by a more aggressive and, in particular, invasive phenotype. SUMMARY: Despite impressively high radiological response rates in patients with recurrent malignant glioma, the duration of response is usually short-lived, and the observed effect to a large extent may be due to normalization of the disrupted blood-brain barrier and less due to a direct antitumor effect. Overall survival remains poor. Induction of invasive phenotypes and escape with proangiogenic alternative pathways are contributing to resistance. Investigation of combination regimes targeting several pathways will determine the possibilities to overcome the resistance to antiangiogenic therapy in malignant gliomas. This article summarizes the results of recent clinical trials in this field, points towards mechanisms of resistance arising under angiogenesis inhibition and discusses the challenges for the future.

Controlled nerve growth factor release from multi-ply alginate/chitosan-based nerve conduits.

The delivery kinetics of growth factors has been suggested to play an important role in the regeneration of peripheral nerves following axotomy. In this context, we designed a nerve conduit (NC) with adjustable release kinetics of nerve growth factor (NGF). A multi-ply system was designed where NC consisting of a polyelectrolyte alginate/chitosan complex was coated with layers of poly(lactide-co-glycolide) (PLGA) to control the release of embedded NGF. Prior to assessing the in vitro NGF release from NC, various release test media, with and without stabilizers for NGF, were evaluated to ensure adequate quantification of NGF by ELISA. Citrate (pH 5.0) and acetate (pH 5.5) buffered saline solutions containing 0.05% Tween 20 yielded the most reliable results for ELISA active NGF. The in vitro release experiments revealed that the best results in terms of reproducibility and release control were achieved when the NGF was embedded between two PLGA layers and the ends of the NC tightly sealed by the PLGA coatings. The release kinetics could be efficiently adjusted by accommodating NGF at different radial locations within the NC. A sustained release of bioactive NGF in the low nanogram per day range was obtained for at least 15days. In conclusion, the developed multi-ply NGF loaded NC is considered a suitable candidate for future implantation studies to gain insight into the relationship between local growth factor availability and nerve regeneration.

L'expérience de la ménopause : étude ethnographique en Suisse Romande et au Centre-Cameroun

Aujourd'hui, la construction sociale de la ménopause intéresse de nombreux chercheurs en sciences sociales. Cependant, la façon dont les femmes vivent ou se représentent cet événement reste peu documentée. L'objectif de cette thèse est donc d'approfondir la compréhension de l'expérience de la ménopause en accordant une place primordiale aux discours et aux pratiques des femmes elles-mêmes. En s'appuyant sur une recherche ethnographique en Suisse romande et au Centre-Cameroun, cette étude parcourt différentes dimensions de l'expérience de la ménopause (représentations, vécus, pratiques de gestion) qui, corrélées les unes aux autres, forment un tout. Au-delà des divergences que l'on peut observer au niveau culturel, de la situation économique et sociale des femmes, des systèmes de santé et du statut occupé par la ménopause dans les deux pays, les résultats de ce travail montrent que les expériences de la ménopause chez les Suissesses et les Camerounaises interviewées ne peuvent pas simplement être classifiées de manière binaire ou dichotomique. Dans chacun des contextes, ces expériences sont plurielles et dépendent de plusieurs facteurs. D'une part, elles découlent d'une observation empirique de la part de ces femmes elles-mêmes et de leurs interactions avec différents acteurs dont les médias (particulièrement en Suisse), les pairs, les proches et les professionnels de la santé. D'autre part, elles sont influencées par de nombreuses variables parmi lesquelles le contexte économique, socioculturel, familial et conjugal, le statut professionnel, la prévalence des troubles ressentis et le statut ménopausique de ces femmes. Mais, ces facteurs ne sont pas hiérarchisés puisqu'ils agissent différemment pour chacune d'entre elles. Dès lors, s'il apparaît que les expériences ménopausiques n'échappent point aux déterminations sociales, il n'en demeure pas moins qu'elles relèvent aussi des capacités réflexives des femmes, les conduites sociales n'étant pas réductibles à des applications des codes intériorisés. Au fil du travail, la médicalisation de la ménopause, bien qu'existant à des degrés variables entre la Suisse et le Cameroun, a émergé comme une problématique transversale. Interrogeant les logiques qui la sous-tendent, cette étude se propose d'analyser le rôle que jouent les femmes elles-mêmes dans ce processus. - Today, the social construction of the menopause is of great interest for many researchers in social sciences. Neverthless, the way of living or of representing this event is still little documented. The aim of this thesis is to study thoroughly the understanding of menopausal experience through the discourses and practices of women themselves. Based on an ethnograph ic research, in French-speaking Switzerland and in Centre-Cameroon, this study looks at different dimensions of menopausal experience (representations, real-life experiences, pratices) that, connected to each other, form a whole. Inspite of the cultural, economic, social, health systems and menopausal status differences between these two countries, the results of this thesis show that menopausal experiences among the Swiss and the Cameroonians interviewee can not simply be classified in two dichotomous groups. In each context, those experiences are plural and depend on several factors. On the one hand, they arise from women's own empirical observations and from their interactions with several actors like the media (especially in Switzerland), the peers, the people closest to them and health professionals. On the other hand, they are influenced by many elements such as the economical, sociocultural, family and marital context, the professional status, the prevalence of felt disorders and the menopausal status of those women. But, these factors are not hierarchical beacause they operate differently for each person. Accordingly, if menopausal experiences don't escape from social determinism, nonetheless they also depend on the reflexive capacities of women beacause social conducts can not be reduced to the application of interiorised codes. Through this work, the médicalisation of the menopause, even though varying between Switzerland and Cameroon, comes to light as a cross-cutting problematic. Questioning its underlying logic, this study proposes to analyse the role that women themselves play in this process.

Natural variability of in vitro adherence to fibrinogen and fibronectin does not correlate with in vivo infectivity of Staphylococcus aureus.

Adherence to fibrinogen and fibronectin plays a crucial role in Staphylococcus aureus experimental endocarditis. Previous genetic studies have shown that infection and carriage isolates do not systematically differ in their virulence-related genes, including genes conferring adherence, such as clfA and fnbA. We set out to determine the range of adherence phenotypes in carriage isolates of S. aureus, to compare the adherence of these isolates to the adherence of infection isolates, and to determine the relationship between adherence and infectivity in a rat model of experimental endocarditis. A total of 133 healthy carriage isolates were screened for in vitro adherence to fibrinogen and fibronectin, and 30 isolates were randomly chosen for further investigation. These 30 isolates were compared to 30 infective endocarditis isolates and 30 blood culture isolates. The infectivities of the carriage isolates, which displayed either extremely low or high adherence to fibrinogen and fibronectin, were tested using a rat model of experimental endocarditis. The levels of adherence to both fibrinogen and fibronectin were very similar for isolates from healthy carriers and members of the two groups of infection isolates. All three groups of isolates showed a wide range of adherence to fibrinogen and fibronectin. Moreover, the carriage isolates that showed minimal adherence and the carriage isolates that showed strong adherence had the same infectivity in experimental endocarditis. Adherence was proven to be important for pathogenesis in experimental endocarditis, but even the least adherent carriage strains had the ability to induce infection. We discuss the roles of differential gene expression, human host factors, and gene redundancy in resolving this apparent paradox.

A novel bioinformatics pipeline to discover genes related to arbuscular mycorrhizal symbiosis based on their evolutionary conservation pattern among higher plants.

BACKGROUND: Genes involved in arbuscular mycorrhizal (AM) symbiosis have been identified primarily by mutant screens, followed by identification of the mutated genes (forward genetics). In addition, a number of AM-related genes has been identified by their AM-related expression patterns, and their function has subsequently been elucidated by knock-down or knock-out approaches (reverse genetics). However, genes that are members of functionally redundant gene families, or genes that have a vital function and therefore result in lethal mutant phenotypes, are difficult to identify. If such genes are constitutively expressed and therefore escape differential expression analyses, they remain elusive. The goal of this study was to systematically search for AM-related genes with a bioinformatics strategy that is insensitive to these problems. The central element of our approach is based on the fact that many AM-related genes are conserved only among AM-competent species. RESULTS: Our approach involves genome-wide comparisons at the proteome level of AM-competent host species with non-mycorrhizal species. Using a clustering method we first established orthologous/paralogous relationships and subsequently identified protein clusters that contain members only of the AM-competent species. Proteins of these clusters were then analyzed in an extended set of 16 plant species and ranked based on their relatedness among AM-competent monocot and dicot species, relative to non-mycorrhizal species. In addition, we combined the information on the protein-coding sequence with gene expression data and with promoter analysis. As a result we present a list of yet uncharacterized proteins that show a strongly AM-related pattern of sequence conservation, indicating that the respective genes may have been under selection for a function in AM. Among the top candidates are three genes that encode a small family of similar receptor-like kinases that are related to the S-locus receptor kinases involved in sporophytic self-incompatibility. CONCLUSIONS: We present a new systematic strategy of gene discovery based on conservation of the protein-coding sequence that complements classical forward and reverse genetics. This strategy can be applied to diverse other biological phenomena if species with established genome sequences fall into distinguished groups that differ in a defined functional trait of interest.

Postpacing abnormal repolarization in catecholaminergic polymorphic ventricular tachycardia associated with a mutation in the cardiac ryanodine receptor gene.

BACKGROUND Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease for which electrophysiological studies (EPS) have shown to be of limited value.OBJECTIVE This study presents a CPVT family in which marked postpacing repolarization abnormalities during EPS were the only consistent phenotypic manifestation of ryanodine receptor (RyR2) mutation carriers.METHODS The study was prompted by the observation of transient marked QT prolongation preceding initiation of ventricular fibrillation during atrial fibrillation in a boy with a family history of sudden cardiac death (SCD). Family members underwent exercise and pharmacologic electrocardiographic testing with epinephrine, adenosine, and flecainide. Noninvasive clinical test results were normal in 10 patients evaluated, except for both epinephrine- and exercise-induced ventricular arrhythmias in 1. EPS included bursts of ventricular pacing and programmed ventricular extrastimulation reproducing short-long sequences. Genetic screening involved direct sequencing of genes involved in long QT syndrome as well as RyR2.RESULTS Six patients demonstrated a marked increase in QT interval only in the first beat after cessation of ventricular pacing and/or extrastimulation. All 6 patients were found to have a heterozygous missense mutation (M4109R) in RyR2. Two of them, presenting with aborted SCD, also had a second missense mutation (I406T- RyR2). Four family members without RyR2 mutations did not display prominent postpacing QT changes.CONCLUSION M4109R- RyR2 is associated with a high incidence of SCD. The contribution of I406T to the clinical phenotype is unclear. In contrast to exercise testing, marked postpacing repolarization changes in a single beat accurately predicted carriers of M4109R- RyR2 in this family.

Circadian variations of renal sodium handling in patients with orthostatic hypotension.

BACKGROUND: Sodium wasting during the night has been postulated as a potential pathophysiological mechanism in patients suffering from orthostatic hypotension due to severe autonomic deficiency. METHODS: In this study, the diurnal variations in creatinine clearance, sodium excretion and segmental renal tubular handling of sodium were evaluated in 18 healthy subjects and 20 young patients with orthostatic hypotension (OH). In addition, 24-hour ambulatory blood pressure and the neuro-hormonal response to changes in posture were determined. The patients and their controls were studied on a free sodium intake. In a second protocol, 10 controls and 10 patients were similarly investigated after one week of a high salt diet (regular diet + 6 g NaCl/day). RESULTS: Our results demonstrate that, in contrast to normal subjects in whom no significant changes in glomerular filtration, sodium excretion and segmental sodium reabsorption were observed throughout the day, patients with OH were characterized by a significant increase in glomerular filtration rate during the nighttime (P = 0.03) and significant increases in urinary lithium excretion (P < 0.05) and lithium clearance (P = 0.05) during the night, suggesting a decreased proximal reabsorption of sodium. On a high sodium diet, the symptoms of orthostatic hypotension and the circadian variations in sodium reabsorption were significantly blunted. CONCLUSIONS: These results suggest that, while the patient is in a supine position the effective blood volume of those with OH becomes excessive due to the increased venous return. Hence, the kidney responds with an increase in glomerular filtration and a relative escape of sodium from the proximal tubular segments. These circadian variations in renal sodium handling may contribute to the maintenance of the orthostatic syndrome.

Gene expression profiling of human glioblastomas for the identification of predictive factors and characterisation of molecular mechanism implicated in response to therapy and outcome

ABSTRACT Poor outcome for glioblastoma patients is largely due to resistance to chemoradiation therapy. While epigenetic inactivation of MGMT mediated DNA repair is highly predictive for benefit from the alkylating agent therapy Temozolomide, additional mechanisms for resistance associated with molecular alterations exist. Furthermore, new concepts in cancer suggest that resistance to treatment may be linked to cancer stem cells that escape therapy and act as source for tumour recurrence. We determined gene expression signatures associated with outcome in glioblastoma patients enrolled in a phase II and phase III clinical trial establishing the new combination therapy of radiation plus concomitant and adjuvant Temozolomide. Correlating stable gene clusters emerging from unsupervised analysis with survival of 42 treated patients identified a number of biological processes associated with outcome. Most prominent, a gene cluster dominated by HOX genes and comprising PROM1, was associated with resistance. PROM1 encodes CD133, a marker for a subpopulation of tumour cells enriched for glioblastoma stem- like cells. The core of this correlated HOX cluster was comprised in the top genes of a "self-renewal signature" defined in a mouse model for MLL-AF9 initiated leukaemia. The association of the HOX gene cluster with tumour resistance was confirmed in two external data sets of 146 malignant glioma As additional resistance factors we identified over-expression of the epidermal growth factor receptor gene, EGFR, while increased gene expression related to biological features of tumour host interaction, including markers for tumour vascular and cell adhesion, and innate immune response, were associated with better outcome. The "self-renewal" signature associated with resistance to the new combination chemoradiation therapy provides first clinical evidence that glioma stem like cells may implicated in resistance in a uniformly treated cohort of glioblastoma patients. This study underlines the need to target the tumour stem cell compartment, and provides some testable hypothesis for biological mechanisms relevant for malignant behaviour of glioblastoma that may be targeted in new treatment approaches. Résumé Le glioblastome, tumeur cérébrale primaire maligne la plus fréquente, est connue pour son mauvais pronostique. Des avancées chimiothérapeutiques récentes avec des agents alkylants comme le témozolomide (TMZ), ont permis une amélioration notable dans la survie de certains patients. Les bénéficiaires ont la caractéristique commune de présenter une particularité génétique, la methylation du MGMT (methylguanine methyltransferase). Néanmoins, d'autres mécanismes de résistance en fonction des aberrations moléculaires existent. Nous avons établi les profils d'expressions génétiques des patients traités par irradiation et TMZ dans des études cliniques de phase II et III. En combinant des méthodes non-supervisées et supervisées, de l'étude de la cohorte des patients traités nous avons découvert des groupes de gènes associés à la survie. Un ensemble de gènes contenant les gènes Hox semble lié au mécanisme de résistance au traitement. Récemment, les gènes Hox ont été décrits comme faisant partie d"une signature d'autorenouvellement (self-renewal) des cellules souches cancéreuses de la leucémie. L'autorenouvellement est un processus grâce auquel les cellules souches se maintiennent tout au long de la vie. Cette association à la résistance est confirmée dans deux autres études indépendantes. Un autre facteur de résistance au traitement est la surexpression du gène EGFR. D'autre part, deux groupes de gènes associés à la relation entre hôte-tumeur tels que les marqueurs des vaisseaux tumoraux et de la réponse immunitaire innée s'avèrent avoir un effet positif sur la survie des patients traités. La découverte de la signature d'autorenouvellement comme facteur de résistance à la nouvelle chimio-radiothérapie offre une preuve clinique que les cellules souches cancéreuses sont impliquées dans la résistance au traitement. If est donc logique de penser que le traitement ciblé contre des cellules souches cancéreuses va dans l'avenir permettre des thérapies anticancéreuses plus performantes.