WW6: an embryonic stem cell line with an inert genetic marker that can be traced in chimeras.

Mutant mice produced by gene targeting in embryonic stem (ES) cells often have a complex or embryonic lethal phenotype. In these cases, it would be helpful to identify tissues and cell types first affected in mutant embryos by following the contribution to chimeras of ES cells homozygous for the mutant allele. Although a number of strategies for following ES cell development in vivo have been reported, each has limitations that preclude its general application. In this paper, we describe ES cell lines that can be tracked to every nucleated cell type in chimeras at all developmental stages. These lines were derived from blastocysts of mice that carry an 11-Mb beta-globin transgene on chromosome 3. The transgene is readily detected by DNA in situ hybridization, providing an inert, nuclear-localized marker whose presence is not affected by transcriptional or translational controls. The "WW" series of ES lines possess the essential features of previously described ES lines, including giving rise to a preponderance of male chimeras, all of which have to date exhibited germ-line transmission. In addition, clones selected for single or double targeting events form strong chimeras, demonstrating the feasibility of using WW6 cells to identify phenotypes associated with the creation of a null mutant.

Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila.

We have used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a vital marker/reporter in Drosophila melanogaster. Transgenic flies were generated in which GFP was expressed under the transcriptional control of the yeast upstream activating sequence that is recognized by GAL4. These flies were crossed to several GAL4 enhancer trap lines, and expression of GFP was monitored in a variety of tissues during development using confocal microscopy. Here, we show that GFP could be detected in freshly dissected ovaries, imaginal discs, and the larval nervous system without prior fixation or the addition of substrates or antibodies. We also show that expression of GFP could be monitored in intact living embryos and larvae and in cultured egg chambers, allowing us to visualize dynamic changes in gene expression during real time.

Insertional mutagenesis and marker rescue in a protozoan parasite: cloning of the uracil phosphoribosyltransferase locus from Toxoplasma gondii.

Nonhomologous integration vectors have been used to demonstrate the feasibility of insertional mutagenesis in haploid tachyzoites of the protozoan parasite Toxoplasma gondii. Mutant clones resistant to 5-fluorouracil were identified at a frequency of approximately 10(-6) (approximately 2 x 10(-5) of the stable transformants). Four independent mutants were isolated, all of which were shown to lack uracil phosphoribosyl-transferase (UPRT) activity and harbor transgenes integrated at closely linked loci, suggesting inactivation of the UPRT-encoding gene. Genomic DNA flanking the insertion point (along with the integrated vector) was readily recovered by bacterial transformation with restriction-digested, self-ligated total genomic DNA. Screening of genomic libraries with the recovered fragment identified sequences exhibiting high homology to known UPRT-encoding genes from other species, and cDNA clones were isolated that contain a single open reading frame predicted to encode the 244-amino acid enzyme. Homologous recombination vectors were exploited to create genetic knock-outs at the UPRT locus, which are deficient in enzyme activity but can be complemented by transient transformation with wild-type sequences--formally confirming identification of the functional UPRT gene. Mapping of transgene insertion points indicates that multiple independent mutants arose from integration at distinct sites within the UPRT gene, suggesting that nonhomologous integration is sufficiently random to permit tagging of the entire parasite genome in a single transformation.

μ-MAR: Multiplane 3D Marker based Registration for depth-sensing cameras

Many applications including object reconstruction, robot guidance, and. scene mapping require the registration of multiple views from a scene to generate a complete geometric and appearance model of it. In real situations, transformations between views are unknown and it is necessary to apply expert inference to estimate them. In the last few years, the emergence of low-cost depth-sensing cameras has strengthened the research on this topic, motivating a plethora of new applications. Although they have enough resolution and accuracy for many applications, some situations may not be solved with general state-of-the-art registration methods due to the signal-to-noise ratio (SNR) and the resolution of the data provided. The problem of working with low SNR data, in general terms, may appear in any 3D system, then it is necessary to propose novel solutions in this aspect. In this paper, we propose a method, μ-MAR, able to both coarse and fine register sets of 3D points provided by low-cost depth-sensing cameras, despite it is not restricted to these sensors, into a common coordinate system. The method is able to overcome the noisy data problem by means of using a model-based solution of multiplane registration. Specifically, it iteratively registers 3D markers composed by multiple planes extracted from points of multiple views of the scene. As the markers and the object of interest are static in the scenario, the transformations obtained for the markers are applied to the object in order to reconstruct it. Experiments have been performed using synthetic and real data. The synthetic data allows a qualitative and quantitative evaluation by means of visual inspection and Hausdorff distance respectively. The real data experiments show the performance of the proposal using data acquired by a Primesense Carmine RGB-D sensor. The method has been compared to several state-of-the-art methods. The results show the good performance of the μ-MAR to register objects with high accuracy in presence of noisy data outperforming the existing methods.

Implications physiopathologiques de la Nestine lors du remodelage pulmonaire et cardiaque à la suite de l’infarctus du myocarde, du diabète et de l’hypertension pulmonaire

Il est reconnu que la protéine filamenteuse intermédiaire Nestine est exprimée lors du processus de cicatrisation et du remodelage fibrotique. De plus, nous avons identifié l’expression de la Nestine au sein de deux populations distinctes qui sont directement impliquées dans les réponses de fibroses réparative et réactive. Ainsi, une population de cellules souches neurales progénitrices résidentes du coeur de rat adulte exprime la Nestine et a été identifiée à titre de substrat de l’angiogenèse et de la neurogenèse cardiaque. Également, la Nestine est exprimée par les myofibroblastes cicatriciels cardiaques et il a été établi que la protéine filamenteuse intermédiaire joue un rôle dans la prolifération de ces cellules. Ainsi, l’objectif général de cette thèse était de mieux comprendre les évènements cellulaires impliqués dans la réponse neurogénique des cellules souches neurales progénitrices résidentes cardiaques Nestine(+) (CSNPRCN(+)) lors de la fibrose réparative cardiaque et d’explorer si l’apparition de fibroblastes Nestine(+) est associée avec la réponse de fibrose réactive secondaire du remodelage pulmonaire. Une première publication nous a permis d’établir qu’il existe une régulation à la hausse de l’expression de la GAP43 (growth associated protein 43) et que cet événement transitoire précède l’acquisition d’un phénotype neuronal par les CSNPRCN(+) lors du processus de cicatrisation cardiaque chez le rat ayant subi un infarctus du myocarde. De plus, la surimposition de la condition diabétique de type 1, via l’injection unique de Streptozotocine chez le rat, abolit la réponse neurogénique des CSNPRCN(+), qui est normalement induite à la suite de l’ischémie cardiaque ou de l’administration de 6-hydroxydopamine. Le second article a démontré que le développement aigu de la fibrose pulmonaire secondaire de l’infarctus du myocarde chez le rat est associé avec une augmentation de l’expression protéique de la Nestine et de l’apparition de myofibroblastes pulmonaires Nestine(+). Également, le traitement de fibroblastes pulmonaires avec des facteurs de croissances peptidiques pro-fibrotiques a augmenté l’expression de la Nestine par ces cellules. Enfin, le développement initial de la condition diabétique de type 1 chez le rat est associé avec une absence de fibrose réactive pulmonaire et à une réduction significative des niveaux protéiques et d’ARN messager de la Nestine pulmonaire. Finalement, la troisième étude représentait quant à elle un prolongement de la deuxième étude et a alors examiné le remodelage pulmonaire chronique chez un modèle établi d’hypertension pulmonaire. Ainsi, les poumons de rats adultes mâles soumis à l’hypoxie hypobarique durant 3 semaines présentent un remodelage vasculaire, une fibrose réactive et une augmentation des niveaux d’ARN messager et de la protéine Nestine. De plus, nos résultats ont démontré que la Nestine, plutôt que l’alpha-actine du muscle lisse, est un marqueur plus approprié des diverses populations de fibroblastes pulmonaires activés. Également, nos données suggèrent que les fibroblastes pulmonaires activés proviendraient en partie de fibroblastes résidents, ainsi que des processus de transition épithélio-mésenchymateuse et de transition endothélio-mésenchymateuse. Collectivement, ces études ont démontré que des populations distinctes de cellules Nestine(+) jouent un rôle majeur dans la fibrose réparative cardiaque et la fibrose réactive pulmonaire.

(Table 2) Stratigraphic marker taxa in ODP Hole 162-985A sediments

The rich and diverse dinocyst assemblages in Cores 162-985A-32X through 62X confirm the importance of these microfossils in unraveling the evolution of the Norwegian Sea. Cosmopolitan taxa, with well-documented stratigraphic ranges in northwest Europe, indicate the following ages: Sections 162-985A-62X-1 through 51X-2, Rupelian (early Oligocene); 50X-5, Oligocene, possibly Chattian; 48X-6, Aquitanian? (early Miocene); 48X-4 through 37X-5, Aquitanian (early Miocene); and 36X-5 through 32X-1, Burdigalian (early Miocene). This stratigraphic interpretation suggests that a major hiatus, which can be correlated with an apparently coeval hiatus at Site 643, occurs within the Chattian at Site 985. Several endemic dinocyst taxa with unusual morphology and restricted stratigraphic occurrences are present in Hole 985A and other Norwegian Sea sites, especially Site 643. By using Hole 985A data for control, the Oligocene-Miocene sediments can be correlated with some degree of confidence in the Norwegian Basin.

(Table T1) Correlations between marker beds and drill holes for ODP Leg 171B sites

More than 50 discrete volcanic ash layers were recovered at the five drill sites of the Blake Nose depth transect (Leg 171B, western central Atlantic). The majority of these ash layers are intercalated with Eocene hemipelagic sediments with a pronounced frequency maximum in the upper Eocene. Several ash layers appear to be deposited from volcanic fallout with little or no indication of secondary remobilization. They provide excellent stratigraphic markers for a correlation of the Leg 171B drill sites. Other ash layers were probably redeposited from volcaniclastic-rich turbidity currents, but they still represent geologically instantaneous events that can be used in stratigraphic correlation between adjacent drill holes. Additional nonvolcanic marker beds, like the suspect late Eocene impact event layer, were included in our hole-to-hole correlations. Stratigraphic and downcore positions of marker beds were compiled and plotted against existing composite depth records that were constructed to guide high-resolution sampling. Comparison of our correlation with the spliced composite sections of each drill site reveals several minor and some major discrepancies. These may result from drilling distortion or missing sections, from the lack of unambiguous criteria for the synchronism of ash layers, or from the systematic exclusion of marker-bed data in the construction of the spliced record. Integration of both correlation approaches will help eliminate most of the observed discrepancies.

Statistical evaluation of the effectiveness of Federal Motor Vehicle Safety Standard 108: Side Marker Lamps (Only). Final report.

National Highway Traffic Safety Administration, Washington, D.C.

Final design and implementation plan for evaluating the effectiveness of FMVSS 108: side marker lamps and high intensity headlamps (only). Tasks #4 and #5 report.

National Highway Traffic Safety Administration, Washington, D.C.

Usage guide for rapid-set epoxy adhesive (118-AF) for traffic marker /

Mode of access: Internet.

Proceedings of the SCAR Conference, held at Langley Research Center, Hampton, Virginia, November, 9-12, 1976. --

Includes bibliographical references.

The scar that tripled; a true story of the great war,

This story relates the same incident told by Richard Harding Davis in The deserter, with a continuation of the narrative.