Molecular characterization and phenotypic expression of mutations in genes for gonadotropins and their receptors in humans.

Gonadotropin hormones undergo important dynamic changes during life. Their rise during puberty stimulates gonadal steroid secretion, triggering the development of secondary sexual characteristics and the acquisition of fertility. The full spectrum of possible mutations and polymorphisms in the human gonadotropins and in their receptor genes has been described in recent years. Patients harboring these mutations display a very wide range of phenotypes affecting all aspects of the reproductive axis. An important insight provided by the careful study of these patients lies in the striking gender differences in the phenotypes associated with a given mutation. As a result, the careful study of these rare patients has allowed us to better define the respective roles of luteinizing hormone and follicle-stimulating hormone in normal human pubertal development and in the achievement of full fertility potential in either males or females. In this work, we describe briefly the known mutations in the genes for both gonadotropins and their receptors, and discuss their genotype/phenotype correlations in light of these important gender differences.

BRX promotes Arabidopsis shoot growth.

? BREVIS RADIX (BRX) has been identified through a loss-of-function allele in the Umkirch-1 accession in a natural variation screen for Arabidopsis root growth vigor. Physiological and gene expression analyses have suggested that BRX is rate limiting for auxin-responsive gene expression by mediating cross-talk with the brassinosteroid pathway, as impaired root growth and reduced auxin perception of brx can be (partially) rescued by external brassinosteroid application. ? Using genetic tools, we show that brx mutants also display significantly reduced cotyledon and leaf growth. ? Similar to the root, the amplitude and penetrance of this phenotype depends on genetic background and shares the physiological features, reduced auxin perception and brassinosteroid rescue. Furthermore, reciprocal grafting experiments between mutant and complemented brx shoot scions and root stocks suggest that the shoot phenotypes are not an indirect consequence of the root phenotype. Finally, BRX gain-of-function lines display epinastic leaf growth and, in the case of dominant negative interference, increased epidermal cell size. Consistent with an impact of BRX on brassinosteroid biosynthesis, this phenotype is accompanied by increased brassinosteroid levels. ? In summary, our results demonstrate a ubiquitous, although quantitatively variable role of BRX in modulating the growth rate in both the root and shoot.

Aggregate cultures of foetal rat liver cells: development and maintenance of liver gene expression.

Rotation-mediated aggregate cultures of foetal rat liver cells were prepared and grown in a chemically defined medium. Their capacity for cellular organisation and maturation was studied over a culture period of 3 wk by using both morphologic and biochemical criteria. It was found that within each aggregate, distinct liver cell types were present and attained their normal, differentiated phenotype. Parenchymal cells formed small acini with a central lumen. Within the first 2 wk in culture, albumin and ferritin mRNA levels were maintained, while the alpha-fetoprotein mRNA levels decreased, and tyrosine aminotransferase (TAT) gene expression increased. No significant response to glucocorticoids was observed in early cultures, whereas after 3 wk a marked increase in TAT mRNA levels was elicited by dexamethasone and glucagon (additive stimulatory effects). The results show that foetal rat liver cells cultured in a chemically defined medium are able to rearrange themselves into histotypic structures, and display a developmental pattern of gene expression comparable to that of perinatal rat liver in vivo. This culture system offers therefore a useful model to study the development and function of liver cells.

Exploring educational mobility in Europe

This paper is concerned with the investigation of the intergenerational mobility of education in several European countries and its changes across birth cohorts (1940-1980) using a new mobility index that considers the total degree of mobility as the weighted sum of mobility with respect to both parents. Moreover, this mobility index enables the analysis of the role of family characteristics as mediating factors in the statistical association between individual and parental education. We find that Nordic countries display lower levels of educational persistence but that the degree of mobility increases over time only in those countries with low initial levels. Moreover, the results suggest that the degree of mobility with respect to fathers and mothers converges to the same level and that family characteristics account for an important part of the statistical association between parental education and children’s schooling; a particular finding is that the most important elements of family characteristics are the family’s socio-economic status and educational assortative mating of the parents.

Transport pathways in the malaria-infected erythrocyte: characterization and their use as potential targets for chemotherapy

The intraerythrocytic malarial parasite is involved in an extremely intensive anabolic activity while it resides in its metabolically quiescent host cell. The necessary fast uptake of nutrients and the discharge of waste product, are guaranteed by parasite-induced alterations of the constitutive transporters of the host cell and the production of new parallel pathways. The membrane of the host cell thus becomes permeable to phospholipids, purine bases and nucleosides, small non-electrolytes, anions and cations. When the new pathways are quantitatively unimportant, classical inhibitors of native transporters can be used to inhibit parasite growth. Several compounds were found to effectively inhibit the new pathways and consequently, parasite growth. The pathways have also been used to introduce cytotoxic agents. The parasitophorous membrane consists of channels which are highly permeable to small solutes and display no ion selectivity. Transport of some cations and anions across the parasite membrane is rapid and insensitive to classical inhibitors, and in some cases it is mediated by specific antiporters which respond to their respective inhibitors. Macromolecules have been shown to reach the parasitophorous space through a duct contiguous with the host cell membrane, and subsequently to be endocytosed at the parasite membrane. The simultaneous presence of the parasitophorous membrane channels and the duct, however, is incompatible with experimental evidences. No specific inhibitors were found as yet that would efficiently inhibit transport through the channels or the duct.

Pyochelin enantiomers and their outer-membrane siderophore transporters in fluorescent pseudomonads: structural bases for unique enantiospecific recognition.

Pyochelin (Pch) and enantiopyochelin (EPch) are enantiomeric siderophores, with three chiral centers, produced under iron limitation conditions by Pseudomonas aeruginosa and Pseudomonas fluorescens , respectively. After iron chelation in the extracellular medium, Pch-Fe and EPch-Fe are recognized and transported by their specific outer-membrane transporters: FptA in P. aeruginosa and FetA in P. fluorescens . Structural analysis of FetA-EPch-Fe and FptA-Pch-Fe, combined with mutagenesis and docking studies revealed the structural basis of the stereospecific recognition of these enantiomers by their respective transporters. Whereas FetA and FptA have a low sequence identity but high structural homology, the Pch and EPch binding pockets do not share any structural homology, but display similar physicochemical properties. The stereospecific recognition of both enantiomers by their corresponding transporters is imposed by the configuration of the siderophore's C4'' and C2'' chiral centers. This recognition involves specific hydrogen bonds between the Arg91 guanidinium group and EPch-Fe for FetA and between the Leu117-Leu116 main chain and Pch-Fe for FptA. FetA and FptA are the first membrane receptors to be structurally described with opposite binding enantioselectivities for their ligands, giving insights into the structural basis of their enantiospecificity.

Estimation of the radiation risks in diagnostic procedures to populations and patients

Since 1895, when X-rays were discovered, ionizing radiation became part of our life. Its use in medicine has brought significant health benefits to the population globally. The benefit of any diagnostic procedure is to reduce the uncertainty about the patient's health. However, there are potential detrimental effects of radiation exposure. Therefore, radiation protection authorities have become strict regarding the control of radiation risks.¦There are various situations where the radiation risk needs to be evaluated. International authority bodies point to the increasing number of radiologic procedures and recommend population surveys. These surveys provide valuable data to public health authorities which helps them to prioritize and focus on patient groups in the population that are most highly exposed. On the other hand, physicians need to be aware of radiation risks from diagnostic procedures in order to justify and optimize the procedure and inform the patient.¦The aim of this work was to examine the different aspects of radiation protection and investigate a new method to estimate patient radiation risks.¦The first part of this work concerned radiation risk assessment from the regulatory authority point of view. To do so, a population dose survey was performed to evaluate the annual population exposure. This survey determined the contribution of different imaging modalities to the total collective dose as well as the annual effective dose per caput. It was revealed that although interventional procedures are not so frequent, they significantly contribute to the collective dose. Among the main results of this work, it was shown that interventional cardiological procedures are dose-intensive and therefore more attention should be paid to optimize the exposure.¦The second part of the project was related to the patient and physician oriented risk assessment. In this part, interventional cardiology procedures were studied by means of Monte Carlo simulations. The organ radiation doses as well as effective doses were estimated. Cancer incidence risks for different organs were calculated for different sex and age-at-exposure using the lifetime attributable risks provided by the Biological Effects of Ionizing Radiations Report VII. Advantages and disadvantages of the latter results were examined as an alternative method to estimate radiation risks. The results show that this method is the most accurate, currently available, to estimate radiation risks. The conclusions of this work may guide future studies in the field of radiation protection in medicine.¦-¦Depuis la découverte des rayons X en 1895, ce type de rayonnement a joué un rôle important dans de nombreux domaines. Son utilisation en médecine a bénéficié à la population mondiale puisque l'avantage d'un examen diagnostique est de réduire les incertitudes sur l'état de santé du patient. Cependant, leur utilisation peut conduire à l'apparition de cancers radio-induits. Par conséquent, les autorités sanitaires sont strictes quant au contrôle du risque radiologique.¦Le risque lié aux radiations doit être estimé dans différentes situations pratiques, dont l'utilisation médicale des rayons X. Les autorités internationales de radioprotection indiquent que le nombre d'examens et de procédures radiologiques augmente et elles recommandent des enquêtes visant à déterminer les doses de radiation délivrées à la population. Ces enquêtes assurent que les groupes de patients les plus à risque soient prioritaires. D'un autre côté, les médecins ont également besoin de connaître le risque lié aux radiations afin de justifier et optimiser les procédures et informer les patients.¦Le présent travail a pour objectif d'examiner les différents aspects de la radioprotection et de proposer une manière efficace pour estimer le risque radiologique au patient.¦Premièrement, le risque a été évalué du point de vue des autorités sanitaires. Une enquête nationale a été réalisée pour déterminer la contribution des différentes modalités radiologiques et des divers types d'examens à la dose efficace collective due à l'application médicale des rayons X. Bien que les procédures interventionnelles soient rares, elles contribuent de façon significative à la dose délivrée à la population. Parmi les principaux résultats de ce travail, il a été montré que les procédures de cardiologie interventionnelle délivrent des doses élevées et devraient donc être optimisées en priorité.¦La seconde approche concerne l'évaluation du risque du point de vue du patient et du médecin. Dans cette partie, des procédures interventionnelles cardiaques ont été étudiées au moyen de simulations Monte Carlo. La dose délivrée aux organes ainsi que la dose efficace ont été estimées. Les risques de développer des cancers dans plusieurs organes ont été calculés en fonction du sexe et de l'âge en utilisant la méthode établie dans Biological Effects of Ionizing Radiations Report VII. Les avantages et inconvénients de cette nouvelle technique ont été examinés et comparés à ceux de la dose efficace. Les résultats ont montré que cette méthode est la plus précise actuellement disponible pour estimer le risque lié aux radiations. Les conclusions de ce travail pourront guider de futures études dans le domaine de la radioprotection en médicine.

Development of Ewing's sarcoma from primary bone marrow-derived mesenchymal progenitor cells.

Ewing's sarcoma is a member of Ewing's family tumors (EFTs) and the second most common solid bone and soft tissue malignancy of children and young adults. It is associated in 85% of cases with the t(11;22)(q24:q12) chromosomal translocation that generates fusion of the 5' segment of the EWS gene with the 3' segment of the ETS family gene FLI-1. The EWS-FLI-1 fusion protein behaves as an aberrant transcriptional activator and is believed to contribute to EFT development. However, EWS-FLI-1 induces growth arrest and apoptosis in normal fibroblasts, and primary cells that are permissive for its putative oncogenic properties have not been discovered, hampering basic understanding of EFT biology. Here, we show that EWS-FLI-1 alone can transform primary bone marrow-derived mesenchymal progenitor cells and generate tumors that display hallmarks of Ewing's sarcoma, including a small round cell phenotype, expression of EFT-associated markers, insulin like growth factor-I dependence, and induction or repression of numerous EWS-FLI-1 target genes. These observations provide the first identification of candidate primary cells from which EFTs originate and suggest that EWS-FLI-1 expression may constitute the initiating event in EFT pathogenesis.

Identification of cancer stem cells in Ewing's sarcoma

Summary : Cancer stem cells (CSC) that display tumor-initiating properties have recently been identified in several distinct types of malignancies, holding promise for more effective therapeutic strategies. However, evidence of such cells in sarcomas, which include some of the most aggressive and therapy-resistant tumors, has not been demonstrated to date. Here, we .identify and characterize cancer stem cells in Ewing's sarcoma family tumors (ESPY), a highly aggressive pediatric malignancy believed to be of mesenchymal stem cell (MSC) origin. Using magnetic bead cell separation of primary ESFT, we have isolated a subpopulation of CD133+ tumor cells that display the capacity to initiate and sustain tumor growth through serial transplantation in NOD/SCID mice, re-establishing at each in vivo passage the parental tumor phenotype and hierarchical cell organization. Consistent with the plasticity of MSCs, in vitro differentiation assays showed that the CD133+ cell population retained the ability to differentiate along adipogenic, osteogenic and chondrogenic lineages. Quantitative Real-Time PCR analysis of genes implicated in stem cell maintenance revealed that CD133+ ESFT cells express significantly higher levels of OCT4 and NANOG than their CD133- counterparts. Taken together, our observations provide the first identification of ESFT cancer stem cells (ET-CSC) and demonstration of their mesenchymal stem cell properties, a critical step toward a better biological understanding and rational therapeutic targeting of these tumors. Résumé : Des cellules souches tumorales avec des propriétés exclusives d'initiation tumorale ont récemment été identifiées dans différents types de cancers, permettant ainsi d'espérer le développement de thérapies plus efficaces. Cependant, l'existence de telles cellules dans les sarcomes, un sous-groupe de cancers d'origine mésenchymateuse très agressifs, n'a pas encore été démontrée. Dans ce travail de recherche, nous identifions et caractérisons des cellules souches tumorales dans le sarcome d'Ewing, une tumeur pédiatrique très agressive vraisemblablement dérivée de cellules souches mésenchymateuses (MSC). Afin de séparer des populations cellulaires dans des échantillons primaires de sarcome d'Ewing, nous avons utilisé des billes magnétiques couplées à des anticorps monoclonaux. Ceci nous a permis d'isoler une sous-population de cellules tumorales CD133+ qui ont la capacité d'initier et de maintenir la croissance tumorale dans des xénotransplantations en série effectuées dans des souris immunodéficientes NOD/SCID. Ces cellules reétablissent à chaque passage in vivo le phénotype de la tumeur d'origine ainsi que son organisation hiérarchique. En accord avec la plasticité des MSC, des tests de différentiation in vitro ont montré que les cellules CD133+ maintiennent la capacité de se différentier en adipocytes, ostéocytes et chondrocytes. Une analyse par PCR quantitative de gènes impliqués dans le maintien des cellules souches a montré que les cellules CD133+ expriment un niveau beaucoup plus élevé de OCT4 and NANOG que les cellules CD133-. En résumé, nos observations constituent la première identification de cellules souches tumorales dans le sarcome d'Ewing et démontrent leur propriété de cellules souches mésenchymateuses. Ceci constitue une étape clé vers une meilleure compréhension biologique et une meilleure approche thérapeutique de ces tumeurs.

Normal glucagon signaling and β-cell function after near-total α-cell ablation in adult mice.

OBJECTIVEEvaluate whether healthy or diabetic adult mice can tolerate an extreme loss of pancreatic α-cells and how this sudden massive depletion affects β-cell function and blood glucose homeostasis.RESEARCH DESIGN AND METHODSWe generated a new transgenic model allowing near-total α-cell removal specifically in adult mice. Massive α-cell ablation was triggered in normally grown and healthy adult animals upon diphtheria toxin (DT) administration. The metabolic status of these mice was assessed in 1) physiologic conditions, 2) a situation requiring glucagon action, and 3) after β-cell loss.RESULTSAdult transgenic mice enduring extreme (98%) α-cell removal remained healthy and did not display major defects in insulin counter-regulatory response. We observed that 2% of the normal α-cell mass produced enough glucagon to ensure near-normal glucagonemia. β-Cell function and blood glucose homeostasis remained unaltered after α-cell loss, indicating that direct local intraislet signaling between α- and β-cells is dispensable. Escaping α-cells increased their glucagon content during subsequent months, but there was no significant α-cell regeneration. Near-total α-cell ablation did not prevent hyperglycemia in mice having also undergone massive β-cell loss, indicating that a minimal amount of α-cells can still guarantee normal glucagon signaling in diabetic conditions.CONCLUSIONSAn extremely low amount of α-cells is sufficient to prevent a major counter-regulatory deregulation, both under physiologic and diabetic conditions. We previously reported that α-cells reprogram to insulin production after extreme β-cell loss and now conjecture that the low α-cell requirement could be exploited in future diabetic therapies aimed at regenerating β-cells by reprogramming adult α-cells.

Behavior of endogenous tumor-associated macrophages assessed in vivo using a functionalized nanoparticle.

Tumor-associated macrophages (TAMs) invade the tumor stroma in many cancers, yet their role is incompletely understood. To visualize and better understand these critical cells in tumor progression, we screened a portfolio of rationally selected, injectable agents to image endogenous TAMs ubiquitously in three different cancer models (colon carcinoma, lung adenocarcinoma, and soft tissue sarcoma). AMTA680, a functionally derivatized magneto-fluorescent nanoparticle, labeled a subset of myeloid cells with an "M2" macrophage phenotype, whereas other neighboring cells, including tumor cells and a variety of other leukocytes, remained unlabeled. We further show that AMTA680-labeled endogenous TAMs are not altered and can be tracked noninvasively at different resolutions and using various imaging modalities, e.g., fluorescence molecular tomography, magnetic resonance imaging, and multiphoton and confocal intravital microscopy. Quantitative assessment of TAM distribution and activity in vivo identified that these cells cluster in delimited foci within tumors, show relatively low motility, and extend cytoplasmic protrusions for prolonged physical interactions with neighboring tumor cells. Noninvasive imaging can also be used to monitor TAM-depleting regimen quantitatively. Thus, AMTA680 or related cell-targeting agents represent appropriate injectable vehicles for in vivo analysis of the tumor microenvironment.

Underwater Gigamosaicing

El procés de fusió de dues o més imatges de la mateixa escena en una d'única i més gran és conegut com a Image Mosaicing. Un cop finalitzat el procés de construcció d'un mosaic, els límits entre les imatges són habitualment visibles, degut a imprecisions en els registres fotomètric i geomètric. L'Image Blending és l'etapa del procediment de mosaicing a la que aquests artefactes són minimitzats o suprimits. Existeixen diverses metodologies a la literatura que tracten aquests problemes, però la majoria es troben orientades a la creació de panorames terrestres, imatges artístiques d'alta resolució o altres aplicacions a les quals el posicionament de la càmera o l'adquisició de les imatges no són etapes rellevants. El treball amb imatges subaquàtiques presenta desafiaments importants, degut a la presència d'scattering (reflexions de partícules en suspensió) i atenuació de la llum i a condicions físiques extremes a milers de metres de profunditat, amb control limitat dels sistemes d'adquisició i la utilització de tecnologia d'alt cost. Imatges amb il·luminació artificial similar, sense llum global com la oferta pel sol, han de ser unides sense mostrar una unió perceptible. Les imatges adquirides a gran profunditat presenten una qualitat altament depenent de la profunditat, i la seva degradació amb aquest factor és molt rellevant. El principal objectiu del treball és presentar dels principals problemes de la imatge subaquàtica, seleccionar les estratègies més adequades i tractar tota la seqüència adquisició-procesament-visualització del procés. Els resultats obtinguts demostren que la solució desenvolupada, basada en una Estratègia de Selecció de Límit Òptim, Fusió en el Domini del Gradient a les regions comunes i Emfatització Adaptativa d'Imatges amb baix nivell de detall permet obtenir uns resultats amb una alta qualitat. També s'ha proposat una estratègia, amb possibilitat d'implementació paral·lela, que permet processar mosaics de kilòmetres d'extensió amb resolució de centímetres per píxel.

Individual contributions of mutant protease and reverse transcriptase to viral infectivity, replication, and protein maturation of antiretroviral drug-resistant human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease (PR) and reverse transcriptase (RT) inhibitors may display impaired infectivity and replication capacity. The individual contributions of mutated HIV-1 PR and RT to infectivity, replication, RT activity, and protein maturation (herein referred to as "fitness") in recombinant viruses were investigated by separately cloning PR, RT, and PR-RT cassettes from drug-resistant mutant viral isolates into the wild-type NL4-3 background. Both mutant PR and RT contributed to measurable deficits in fitness of viral constructs. In peripheral blood mononuclear cells, replication rates (means +/- standard deviations) of RT recombinants were 72.5% +/- 27.3% and replication rates of PR recombinants were 60.5% +/- 33.6% of the rates of NL4-3. PR mutant deficits were enhanced in CEM T cells, with relative replication rates of PR recombinants decreasing to 15.8% +/- 23.5% of NL4-3 replication rates. Cloning of the cognate RT improved fitness of some PR mutant clones. For a multidrug-resistant virus transmitted through sexual contact, RT constructs displayed a marked infectivity and replication deficit and diminished packaging of Pol proteins (RT content in virions diminished by 56.3% +/- 10.7%, and integrase content diminished by 23.3% +/- 18.4%), a novel mechanism for a decreased-fitness phenotype. Despite the identified impairment of recombinant clones, fitness of two of the three drug-resistant isolates was comparable to that of wild-type, susceptible viruses, suggestive of extensive compensation by genomic regions away from PR and RT. Only limited reversion of mutated positions to wild-type amino acids was observed for the native isolates over 100 viral replication cycles in the absence of drug selective pressure. These data underscore the complex relationship between PR and RT adaptive changes and viral evolution in antiretroviral drug-resistant HIV-1.

Expression of substance P and preprotachykinin mRNA by primary sensory neurons in culture: regulation by factors present in peripheral and central target tissues.

Primary sensory neurons display various neuronal phenotypes which may be influenced by factors present in central or peripheral targets. In the case of DRG cells expressing substance P (SP), the influence of peripheral or central targets was tested on the neuronal expression of this neuropeptide. DRG cells were cultured from chick embryo at E6 or E10 (before or after establishment of functional connections with targets). Preprotachykinin mRNA was visualized in DRG cell cultures by either Northern blot or in situ hybridization using an antisense labeled riboprobe, while the neuropeptide SP was detected by immunostaining with a monoclonal antibody. In DRG cell cultures from E10, only 60% of neurons expressed SP. In contrast, DRG cell cultures performed at E6 showed a significant hybridization signal and SP-like immunoreactivity in virtually all the neurons (98%). The addition of extracts from muscle, skin, brain or spinal cord to DRG cells cultured at E6 reduced by 20% the percentage of neurons which express preprotachykinin mRNA and SP-like immunoreactivity. Our results indicate that factors issued from targets inhibit SP-expression by a subset of primary sensory neurons and act on the transcriptional control of preprotachykinin gene.

Spondylarthropathies in children--are they different from those in adults?

Juvenile spondylarthropathies (JSpAs) comprise a group of rheumatic diseases distinct from other categories of juvenile arthritis. Several classification systems have been applied, and some are specific for children, such as the seronegative enthesopathy and arthropathy (SEA) syndrome and the enthesitis-related arthritis, diagnostic forms in the International League of Associations for Rheumatism (ILAR) classification. JSpA seems more frequent than was previously believed, but actual epidemiological data show important variations between studies. Compared to adult patients, children with JSpA present with peripheral arthritis and enthesitis early in disease but sacroiliac and spine joints involvement many years later. A multidisciplinary team in a paediatric environment should be responsible for the management of children with spondylarthropathies to ensure the best care for these children with their chronic disease and risk of long-term disability. Recent advances in the treatment of rheumatic diseases with biological agents show promising results in children with JSpA. Further research needs to be conducted to increase our knowledge of the long-term outcome of these patients, to improve management, and to prevent long-term consequences of the disease.