Antigenic heterogeneity of clones and subclones from human melanoma cell lines demonstrated by a panel of monoclonal antibodies and flow microfluorometry analysis.

Cells from two melanoma cell lines, Me43 and GLL-19, were cloned in methylcellulose cultures and 20 randomly selected colonies from each line were picked up by micromanipulation, expanded in liquid cultures, and considered as clones of the original cell lines. The antigenic cell surface phenotype of these clones defined by panel of 12 monoclonal antibodies (MAb) was analyzed by flow microfluorometry (FMF) using a fluorescence-activated cell sorter (FACS II) and compared with the known stable phenotype of the parent cell line. The antibody panel consisted of eight MAb against melanoma-associated antigens, two MAb against monomorphic determinants of HLA-DR (la) and HLA-ABC, respectively, one MAb against the common acute lymphoblastic leukemia antigen (CALLA) and one MAb against carcinoembryonic antigen used as control. A remarkable heterogeneity in terms of qualitative and quantitative expression of the cell surface antigens studied was observed among and within the different clones. The single-cell origin of the clones was assessed by comparing the clonogenic cell frequency, determined by limiting dilutions in microculture plates, with the cloning efficiency observed in Petri dishes. Both techniques using methylcellulose medium gave the same percentages of growing colonies. Cells from four Me43 clones were recloned in methylcellulose and the phenotype of five randomly selected subclones from each clone was analysed using the same panel of monoclonal antibodies. Each subclone also displayed heterogeneity with individual phenotypes different from that of the original clone and from the parental Me43 cell line. The antigen expression by individual cells in situ within clones was analyzed on frozen sections from colonies using the same panel of MAb and a biotin-avidin immunoperoxidase method. The results confirmed the marked heterogeneity of antigen expression within and among colonies, as indicated by the FMF analysis.

A genome-wide significant linkage for severe depression on chromosome 3: the depression network study.

Objective: The purpose of this study was to find loci for major depression via linkage analysis of a large sibling pair sample. Method: The authors conducted a genome-wide linkage analysis of 839 families consisting of 971 affected sibling pairs with severe recurrent major depression, comprising waves I and II of the Depression Network Study cohort. In addition to examining affected status, linkage analyses in the full data set were performed using diagnoses restricted by impairment severity, and association mapping of hits in a large case-control data set was attempted. Results: The authors identified genome-wide significant linkage to chromosome 3p25-26 when the diagnoses were restricted by severity, which was a maximum LOD score of 4.0 centered at the linkage marker D3S1515. The linkage signal identified was genome-wide significant after correction for the multiple phenotypes tested, although subsequent association mapping of the region in a genome-wide association study of a U.K. depression sample did not provide significant results. Conclusions: The authors report a genome-wide significant locus for depression that implicates genes that are highly plausible for involvement in the etiology of recurrent depression. Despite the fact that association mapping in the region was negative, the linkage finding was replicated by another group who found genome-wide-significant linkage for depression in the same region. This suggests that 3p25-26 is a new locus for severe recurrent depression. This represents the first report of a genome-wide significant locus for depression that also has an independent genome-wide significant replication.

Occurrence of hybrids and laboratory evidence of fertility among three species of the Phyllosoma complex (Hemiptera: Reduviidae) in Mexico

In seven studied communities of Western Mexico, triatomine specimens were sympatrically collected, some with atypical morphological characteristics in contrast to pure specimens, which were presumed to be hybrids. More than 200 specimens of Meccus pallidipennis and Meccus longipennis with brown-yellow markings on dorsal connexival segments were collected in Ahuacapán and Quitupan. In La Mesa, more than 60 specimens similar to Meccus picturatus in most morphological characteristics (including size) were collected, although they presented a largely yellowish corium like M. pallidipennis. Interfertility was proven between all of the studied wild hybrid specimens, as well as between all the experimental laboratory hybrids. Two different phenotypes (M. picturatus and M. longipennis) were obtained from crosses between M. picturatus x M. picturatus and M. longipennis x M. longipennis from the three studied localities in state of Nayarit as from La Mesita. Results support the hypothesis that the subspecific ranking of those triatomines may, therefore, be more appropriate because reproductive isolation has not been developed and complete interbreeding was recorded.

Semi-online neural-Q_leaming for real-time robot learning

Reinforcement learning (RL) is a very suitable technique for robot learning, as it can learn in unknown environments and in real-time computation. The main difficulties in adapting classic RL algorithms to robotic systems are the generalization problem and the correct observation of the Markovian state. This paper attempts to solve the generalization problem by proposing the semi-online neural-Q_learning algorithm (SONQL). The algorithm uses the classic Q_learning technique with two modifications. First, a neural network (NN) approximates the Q_function allowing the use of continuous states and actions. Second, a database of the most representative learning samples accelerates and stabilizes the convergence. The term semi-online is referred to the fact that the algorithm uses the current but also past learning samples. However, the algorithm is able to learn in real-time while the robot is interacting with the environment. The paper shows simulated results with the "mountain-car" benchmark and, also, real results with an underwater robot in a target following behavior

Coordinated effects of sequence variation on DNA binding, chromatin structure, and transcription.

DNA sequence variation has been associated with quantitative changes in molecular phenotypes such as gene expression, but its impact on chromatin states is poorly characterized. To understand the interplay between chromatin and genetic control of gene regulation, we quantified allelic variability in transcription factor binding, histone modifications, and gene expression within humans. We found abundant allelic specificity in chromatin and extensive local, short-range, and long-range allelic coordination among the studied molecular phenotypes. We observed genetic influence on most of these phenotypes, with histone modifications exhibiting strong context-dependent behavior. Our results implicate transcription factors as primary mediators of sequence-specific regulation of gene expression programs, with histone modifications frequently reflecting the primary regulatory event.

Vaccinia virus regulates expression of p21WAF1/Cip1 in A431 cells

In this paper, we provide evidence that both the mRNA and protein levels of the cyclin-dependent kinase (CDK) inhibitor p21WAF1/CDK-interacting protein 1 (Cip1) increase upon infection of A431 cells with Vaccinia virus (VACV). In addition, the VACV growth factor (VGF) seems to be required for the gene expression because infection carried out with the mutant virus VACV-VGF- revealed that this strain was unable to stimulate its transcription. Our findings are also consistent with the notion that the VGF-mediated change in p21WAF1/Cip1 expression is dependent on tyrosine kinase pathway(s) and is partially dependent on mitogen-activated protein kinase/extracellular-signal regulated kinase 1/2. We believe that these pathways are biologically significant because VACV replication and dissemination was drastically affected when the infection was carried out in the presence of the relevant pharmacological inhibitors.

Molecular analysis of non-melanoma skin cancer

SUMMARY:Cylindroma, trichoepithelioma and spiradenoma are benign tumors of hair follicle. They are caused by mutations and loss of heterozygosity in the CYLD gene. CYLD is a ubiquitously expressed, but the tumors are restricted to skin, suggesting that the tumorigenesis is influenced by skin-specific regulators and probably by mutations in other genes. The objectives of the thesis were to analyze the molecular mechanisms leading to the aforementioned tumors. In the first project, we have identified five new mutations in CYLD gene in tive families affected with different combinations of these skin appendage tumors. F our of these mutations caused the introduction of a premature stop codon in CYLD protein sequence, but one was a missense mutation changing aspartic acid 681 into glycine (D68lG), in patients exhibiting multiple trichoepitheliomas. CYLD is a deubiquitinase which can downregulate NF-κB and INK pathways through the deubiquitination of TRAF2, for example. We showed that the CYLD-D681G mutant was unable to remove polyubiquitin chains from TRAF2. We also proved that CYLD-D68lG could not inhibit TRAP 2- or TNFα- mediated NF-κB or INK activations in 293T cells. These results underlined the importance of the D68l residue for the enzymatic activity of CYLD. TRAP-interacting protein (TRIP), which is a E3-Ubiquitin ligase, is a partner of CYLD. In the second project of the thesis, we studied the function of TRIP in the epidermis. We found that TRIP was a nucleolar protein in cultured human primary keratinocytes (HEK) and HeLa cells, and was detected in the midbody of HeLa cells. Moreover, TRIP expression was shown to be downregulated through a PKC-dependent mechanism before induction of keratinocyte differentiation. We also proved that TRIP was upregulated in basal cell carcinomas. Furthermore, TRIP was found to be important for keratinocyte survival and proliferation through the regulation of the Gl/S transition. Our results suggest that TRIP may be involved in keratinocyte tumorigenesis.RÉSUMÉ :Les cylindromes, trichoépithéliomes et spiradénomes sont des tumeurs bénignes du follicule pileux causées par des mutations et une perte d'hétérozygotie du gène CYLD. CYLD est ubiquitaire mais les tumeurs sont limitées à la peau, suggérant que la tumorigénèse est influencée par des protéines spécifiques de la peau et par des mutations dans d'autres gènes. Les objectifs de la thèse étaient d'2malyser les mécanismes moléculaires aboutissant à la formation de ces tumeurs. Dans le premier projet, cinq nouvelles mutations du gène CYLD ont été identifiées chez cinq familles présentant différentes combinaisons des tumeurs citées ci- dessus. Quatre de ces mutations causaient I' introduction d'un codon stop prématuré dans la séquence protéique, mais une était une mutation «misser1se» changeant l'aspartate 681 en résidu glycine (D68lG) chez des patients présentant des trichoépithéliomes multiples. CYLD est une déubiquitinase qui inhibe les voies de signalisation de NF-κB et JNK, en déubiquitinant notamment TRAF2. Nous avons montré que la protéine mutante CYLD- D68lG ne pouvait pas cliver la chaîne de poly-ubiquitines liée à TRAF2. CYLD-D68lG était aussi incapable d'inhiber l'activation de NF-κB ou de JNK induite par TRAF2 ou TNF-o dans les cellules 293T. Ces résultats ont donc souligné l'impo1tance du résidu D68l pour l'activité de CYLD. «TRAF-interacting protein (TRIP)», qui est une «E3-ubiquitin-ligase», est un partenaire de CYLD. Dans le second proj et de la thèse, nous avons étudié la fonction de TRIP dans l'épidenne. Nous avons montrépque TRIP était nucléolaire dans les cellules HeLa et les kératinocytes primaires humains en culture et était détectée dans le «midbody» des cellules HeLa. Nous avons prouvé que l'ARNm de TRIP était diminué avant l'induction de la différentiation des kératinocytes, par un mécanisme dépendent de la protéine kinase C, tandis qu'il était augmenté dans les carcinomes baso-cellulaires. Nous avons aussi montré que TRIP influençait la prolifération et la survie des kératinocytes en régulant la transition G1/S, Nos résultats suggèrent que TRIP est peut-être impliquée dans la tumorigénèse des kératinocytes. 7

Confabulation in schizophrenia: a neuropsychological study.

Confabulation has been documented in schizophrenia, but its neuropsychological correlates appear to be different from those of confabulation in neurological disease states. Forty-five schizophrenic patients and 37 controls were administered a task requiring them to recall fables. They also underwent testing with a range of memory and executive tasks. The patients with schizophrenia produced significantly more confabulations than the controls. After correcting for multiple comparisons, confabulation was not significantly associated with memory impairment, and was associated with impairment on only one of eight executive measures, the Brixton Test. Confabulation scores were also associated with impairment on two semantic memory tests. Confabulation was correlated with intrusion errors in recall, but not false positive errors in a recognition task. The findings suggest that confabulation in schizophrenia is unrelated to the episodic memory impairment seen in the disorder. However, the association with a circumscribed deficit in executive function could be consistent with a defective strategic retrieval account of confabulation similar to that of Moscovitch and co-workers, interacting with defective semantic memory.

Role of Notch signaling in the skin and cornea

Résumé : La voie de signalisation Notch est essentielle pour la différentiation de l'épiderme lors du développement embryonnaire de la peau. Il a été démontré que l'inactivation de Notch1 dans la peau de souris conduit à une hyperplasie de l'épiderme ainsi qu'à la formation subséquente de carcinomes basocellulaires ainsi que de plaques cornéennes. L'inactivation de Notch1 dans la cornée combinée à des lésions mécaniques démontre que les cellules progénitrices de la cornée se différentient en un épithélium hyperplasique et kératinisé comme la peau. Ce changement de destinée cellulaire conduit à une cécité cornéenne et implique des processus non-autonomes aux cellules épithéliales, caractérisés par la sécrétion de FGF-2 par l'épithélium Notch1-/- suivi d'une vascularisation et d'un remaniement du stroma sous-jacent. La déficience en vitamine A est connu comme cause de lésions cornéennes humaines (xérophtalmie sévère). En accord, nous avons trouvé que la signalisation Notch1 était liée au métabolisme de la vitamine A par la régulation de l'expression de CRBP1, nécessaire pour générer un pool de rétinol intracellulaire. La perte de Notch1 dans l'épiderme, l'autre récepteur de la famille présent dans la peau marine, ne conduit pas à un phénotype manifeste. Cependant, l'inactivation dans l'épiderme de Notch1 et Notch2 ensemble, ou de RBP-J, induit une dermatite atopique (DA) sévère chez les souris. De même, les patients souffrants de DA mais pas ceux souffrant de psoriasis ou de lichen plan, ont une réduction marquée de l'expression des récepteurs Notch dans la peau. La perte de Notch dans les keratinocytes conduit à une activation de la voie NF-κB, ce qui ensuite induit la production de TSLP, une cytokine profondément impliquée dans la pathogenèse de la DA. Nous démontrons génétiquement que TSLP est responsable de la DA ainsi que du développent d'un syndrome myéloprolifératif non-autonome aux cellules induit par le G-CSF. Cependant, ces souris avec une inactivation dans l'épiderme de Notch1 et Notch2 et aussi incapables de répondre au TSLP développent des tumeurs invasive sévères caractérisées par une haute activité de signalisation ß-catenin. TSLPR est identifié comme un potentiel suppresseur de tumeur non-autonome aux cellules tumorales; la transplantation de cellules hématopoïétiques TSLPR-/- dans des souris déficientes pour Notch est suffisant pour causer des tumeurs. Summary : The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. It has previously been demonstrated that Notch1 inactivation in marine skin results in epidermal hyperplasia and subsequent formation of basal cell carcinoma-like (BCC-like) tumors as well as corneal plaques. Inducible ablation of Notch1 in the cornea combined with mechanical wounding show that Notch1 deficient corneal progenitor cells differentiate into a hyperplasic, keratinized, skin-like epithelium. This cell fate switch leads to corneal blindness and involves cell non-autonomous processes, characterized by secretion of FGF-2 through Notch1-/- epithelium followed by vascularisation and remodelling of the underlying stroma. Vitamin A deficiency is known to induce a similar corneal defect in humans (severe xerophthalmia). Accordingly, we found that Notch1 signaling is linked to vitamin A metabolism by regulating the expression of CRBP1, required to generate a pool of intracellular retinol. Epidermal loss of Notch2, the other Notch receptor present in marine skin, doesn't lead to any overt phenotypes. However, postnatal epidermis-specific inactivation of both Notch1 and Notch2, or of RBP-J, induces the development of a severe form of atopic dermatitis (AD) in mice. Likewise, patients suffering from AD, but not psoriasis or lichen planas, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes leads to an activation of NF-κB signaling which in turn induces the production of Thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. We genetically demonstrate that TSLP is responsible for AD as well as the development of a cell non-autonomous G-CSF induced myeloproliferative disorder (MPD) in mice. However, these mice with conditional epidermal inactivation of Notch1 and Notch2 as well as incapable to respond to TSLP develop severe invasive tumors characterized by high ß-catenin signaling activity. TSLPR is identified as a potential cell non-autonomous tumor suppressor; transplantation of TSLPR-/- hematopoietic cells into epidermal Notch deficient mice is sufficient to cause tumors.

Bovine tuberculosis in Doñana Biosphere Reserve: the role of wild ungulates as disease reservoirs in the last Iberian lynx strongholds.

Doñana National Park (DNP) in southern Spain is a UNESCO Biosphere Reserve where commercial hunting and wildlife artificial feeding do not take place and traditional cattle husbandry still exists. Herein, we hypothesized that Mycobacterium bovis infection prevalence in wild ungulates will depend on host ecology and that variation in prevalence will reflect variation in the interaction between hosts and environmental risk factors. Cattle bTB reactor rates increased in DNP despite compulsory testing and culling of infected animals. In this study, 124 European wild boar, 95 red deer, and 97 fallow deer were sampled from April 2006 to April 2007 and analyzed for M. bovis infection. Modelling and GIS were used to identify risk factors and intra and inter-species relationships. Infection with M. bovis was confirmed in 65 (52.4%) wild boar, 26 (27.4%) red deer and 18 (18.5%) fallow deer. In the absence of cattle, wild boar M. bovis prevalence reached 92.3% in the northern third of DNP. Wild boar showed more than twice prevalence than that in deer (p<0.001). Modelling revealed that M. bovis prevalence decreased from North to South in wild boar (p<0.001) and red deer (p<0.01), whereas no spatial pattern was evidenced for fallow deer. Infection risk in wild boar was dependent on wild boar M. bovis prevalence in the buffer area containing interacting individuals (p<0.01). The prevalence recorded in this study is among the highest reported in wildlife. Remarkably, this high prevalence occurs in the absence of wildlife artificial feeding, suggesting that a feeding ban alone would have a limited effect on wildlife M. bovis prevalence. In DNP, M. bovis transmission may occur predominantly at the intra-species level due to ecological, behavioural and epidemiological factors. The results of this study allow inferring conclusions on epidemiological bTB risk factors in Mediterranean habitats that are not managed for hunting purposes. Our results support the need to consider wildlife species for the control of bTB in cattle and strongly suggest that bTB may affect animal welfare and conservation.

Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition.

BACKGROUND ErbB2-positive breast cancer is characterized by highly aggressive phenotypes and reduced responsiveness to standard therapies. Although specific ErbB2-targeted therapies have been designed, only a small percentage of patients respond to these treatments and most of them eventually relapse. The existence of this population of particularly aggressive and non-responding or relapsing patients urges the search for novel therapies. The purpose of this study was to determine whether cannabinoids might constitute a new therapeutic tool for the treatment of ErbB2-positive breast tumors. We analyzed their antitumor potential in a well established and clinically relevant model of ErbB2-driven metastatic breast cancer: the MMTV-neu mouse. We also analyzed the expression of cannabinoid targets in a series of 87 human breast tumors. RESULTS Our results show that both Delta9-tetrahydrocannabinol, the most abundant and potent cannabinoid in marijuana, and JWH-133, a non-psychotropic CB2 receptor-selective agonist, reduce tumor growth, tumor number, and the amount/severity of lung metastases in MMTV-neu mice. Histological analyses of the tumors revealed that cannabinoids inhibit cancer cell proliferation, induce cancer cell apoptosis, and impair tumor angiogenesis. Cannabinoid antitumoral action relies, at least partially, on the inhibition of the pro-tumorigenic Akt pathway. We also found that 91% of ErbB2-positive tumors express the non-psychotropic cannabinoid receptor CB2. CONCLUSIONS Taken together, these results provide a strong preclinical evidence for the use of cannabinoid-based therapies for the management of ErbB2-positive breast cancer.

Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study

Enteroinvasive Escherichia coli (EIEC) and Shigellaspp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigellashare many genetic and biochemical similarities, the illness caused by Shigellais more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneriwith cultured J774 macrophage-like cells. We evaluated several phenotypes: (i) bacterial escape from macrophages after phagocytosis, (ii) macrophage death induced by EIEC and S. flexneri, (iii) macrophage cytokine expression in response to infection and (iv) expression of plasmidial (pINV) virulence genes. The results showed thatS. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.

Embodied Autonomy in Digital Ecosystems: From Bio-Inspired Agents to Cognitive Systems

This paper proposes to promote autonomy in digital ecosystems so that it provides agents with information to improve the behavior of the digital ecosystem in terms of stability. This work proposes that, in digital ecosystems, autonomous agents can provide fundamental services and information. The final goal is to run the ecosystem, generate novel conditions and let agents exploit them. A set of evaluation measures must be defined as well. We want to provide an outline of some global indicators, such as heterogeneity and diversity, and establish relationships between agent behavior and these global indicators to fully understand interactions between agents, and to understand the dependence and autonomy relations that emerge between the interacting agents. Individual variations, interaction dependencies, and environmental factors are determinants of autonomy that would be considered. The paper concludes with a discussion of situations when autonomy is a milestone

Parental influences on pathogen resistance in brown trout embryos and effects of outcrossing within a river network.

Phenotypic plasticity can increase tolerance to heterogeneous environments but the elevations and slopes of reaction norms are often population specific. Disruption of locally adapted reaction norms through outcrossing can lower individual viability. Here, we sampled five genetically distinct populations of brown trout (Salmo trutta) from within a river network, crossed them in a full-factorial design, and challenged the embryos with the opportunistic pathogen Pseudomonas fluorescens. By virtue of our design, we were able to disentangle effects of genetic crossing distance from sire and dam effects on early life-history traits. While pathogen infection did not increase mortality, it was associated with delayed hatching of smaller larvae with reduced yolk sac reserves. We found no evidence of a relationship between genetic distance (W, FST) and the expression of early-life history traits. Moreover, hybrids did not differ in phenotypic means or reaction norms in comparison to offspring from within-population crosses. Heritable variation in early life-history traits was found to remain stable across the control and pathogen environments. Our findings show that outcrossing within a rather narrow geographical scale can have neutral effects on F1 hybrid viability at the embryonic stage, i.e. at a stage when environmental and genetic effects on phenotypes are usually large.

Interplay between insulin signaling, juvenile hormone, and vitellogenin regulates maternal effects on polyphenism in ants.

Polyphenism is the phenomenon in which alternative phenotypes are produced by a single genotype in response to environmental cues. An extreme case is found in social insects, in which reproductive queens and sterile workers that greatly differ in morphology and behavior can arise from a single genotype. Experimental evidence for maternal effects on caste determination, the differential larval development toward the queen or worker caste, was recently documented in Pogonomyrmex seed harvester ants, in which only colonies with a hibernated queen produce new queens. However, the proximate mechanisms behind these intergenerational effects have remained elusive. We used a combination of artificial hibernation, hormonal treatments, gene expression analyses, hormone measurements, and vitellogenin quantification to investigate how the combined effect of environmental cues and hormonal signaling affects the process of caste determination in Pogonomyrmex rugosus. The results show that the interplay between insulin signaling, juvenile hormone, and vitellogenin regulates maternal effects on the production of alternative phenotypes and set vitellogenin as a likely key player in the intergenerational transmission of information. This study reveals how hibernation triggers the production of new queens in Pogonomyrmex ant colonies. More generally, it provides important information on maternal effects by showing how environmental cues experienced by one generation can translate into phenotypic variation in the next generation.