High frequencies of leukemia stem cells in poor-outcome childhood precursor-B acute lymphoblastic leukemias.

In order to develop a xenograft model to determine the efficacy of new therapies against primary human precursor-B acute lymphoblastic leukemia (ALL) stem cells (LSCs), we used the highly immunodeficient non-obese diabetic (NOD).Cg-Prkdc(scid)IL2rg(tmlWjl)/SzJ (NOD-severe combined immune deficient (scid) IL2rg(-/-)) mouse strain. Intravenous transplantation of 2 of 2 ALL cell lines and 9 of 14 primary ALL cases generated leukemia-like proliferations in recipient mice by 1-7 months after transplant. Leukemias were retransplantable, and the immunophenotypes, gene rearrangements and expression profiles were identical or similar to those of the original primary samples. NOD-scid mice transplanted with the same primary samples developed similar leukemias with only a slightly longer latency than did NOD-scid-IL2Rg(-/-) mice. In this highly sensitive NOD-scid-IL2Rg(-/-)-based assay, 1-100 unsorted primary human ALL cells from five of five tested patients, four of whom eventually experienced leukemia relapse, generated leukemias in recipient mice. This very high frequency of LSCs suggests that a hierarchical LSC model is not valuable for poor-outcome ALL.

Spidey: a tool for mRNA-to-genomic alignments.

We have developed a computer program that aligns spliced sequences to genomic sequences, using local alignment algorithms and heuristics to put together a global spliced alignment. Spidey can produce reliable alignments quickly, even when confronted with noise from alternative splicing, polymorphisms, sequencing errors, or evolutionary divergence. We show how Spidey was used to align reference sequences to known genomic sequences and then to the draft human genome, to align mRNAs to gene clusters, and to align mouse mRNAs to human genomic sequence. We compared Spidey to two other spliced alignment programs; Spidey generally performed quite well in a very reasonable amount of time.

Sequence and comparative analysis of the mouse 1-megabase region orthologous to the human 11p15 imprinted domain.

A major barrier to conceptual advances in understanding the mechanisms and regulation of imprinting of a genomic region is our relatively poor understanding of the overall organization of genes and of the potentially important cis-acting regulatory sequences that lie in the nonexonic segments that make up 97% of the genome. Interspecies sequence comparison offers an effective approach to identify sequence from conserved functional elements. In this article we describe the successful use of this approach in comparing a approximately 1-Mb imprinted genomic domain on mouse chromosome 7 to its orthologous region on human 11p15.5. Within the region, we identified 112 exons of known genes as well as a novel gene identified uniquely in the mouse region, termed Msuit, that was found to be imprinted. In addition to these coding elements, we identified 33 CpG islands and 49 orthologous nonexonic, nonisland sequences that met our criteria as being conserved, and making up 4.1% of the total sequence. These conserved noncoding sequence elements were generally clustered near imprinted genes and the majority were between Igf2 and H19 or within Kvlqt1. Finally, the location of CpG islands provided evidence that suggested a two-island rule for imprinted genes. This study provides the first global view of the architecture of an entire imprinted domain and provides candidate sequence elements for subsequent functional analyses.

Domain size distributions can predict domain boundaries.

MOTIVATION: The sizes of protein domains observed in the 3D-structure database follow a surprisingly narrow distribution. Structural domains are furthermore formed from a single-chain continuous segment in over 80% of instances. These observations imply that some choices of domain boundaries on an otherwise uncharacterized sequence are more likely than others, based solely on the size and segment number of predicted domains. This property might be used to guess the locations of protein domain boundaries. RESULTS: To test this possibility we enumerate putative domain boundaries and calculate their relative likelihood under a probability model that considers only the size and segment number of predicted domains. We ask, in a cross-validated test using sequences with known 3D structure, whether the most likely guesses agree with the observed domain structure. We find that domain boundary predictions are surprisingly successful for sequences up to 400 residues long and that guessing domain boundaries in this way can improve the sensitivity of threading analysis.

Human and nematode orthologs--lessons from the analysis of 1800 human genes and the proteome of Caenorhabditis elegans.

Recently, we have defined and analyzed over 1800 orthologous human and rodent genes. Here we extend this work to compare human and Caenorhabditis elegans coding sequences. 1880 human proteins were compared with about 20000 predicted nematode proteins presumably comprising nearly the complete proteome of C. elegans. We found that 44% of human/rodent orthologs have convincing nematode counterparts. On average, the amino acid similarity and identity between aligned human and C. elegans orthologous gene products are 69.3% and 49.1% respectively, and the nucleotide identity is 49.8%. Detailed investigation of our results suggests that some nematode gene predictions are incorrect, leading to erroneous pairing with human genes (e.g. calcineurin and polymerase II elongation factor III). Furthermore, other proteins (i.e. homologs of human ribosomal proteins S20 and L41, thymosin) are missing entirely from the nematode proteome, suggesting that it may not be complete. These results underscore the fact that metazoan gene prediction is a very challenging task and that most computer-predicted nematode genes require supporting evidence of their existence from comparative genomics and/or laboratory investigation.

Take teamwork to new heights.

Teamwork follows the dynamic path of group development. The authors evaluate their survey of teamwork in 14 ICUs and explain how nurse managers can evaluate and increase teamwork with a unit assessment process.

Assessing the functional level of rehabilitation teams and facilitating team development.

This article describes a research-based method for assessing team effectiveness and for facilitating the development of rehabilitation teams. The authors used a 60-item self-report instrument to measure the developmental level of a group or a team. Two examples of the use of this instrument with rehabilitation teams are discussed.

Species differences in responses to hyperosmolality and D600 in cat and rat heart.

The direct inotropic effect of hypertonic mannitol was compared in isolated rat and cat papillary muscles. The inotropic effects of paired electrical stimulation and D600 were also evaluated in the same species. At extracellular calcium concentrations of 2.5 mM, hypertonic mannitol (25--100 mosmol/kg H2O above normal) depressed contractility in isolated rat myocardium; hyperosmolality exerted a positive effect only when extracellular Ca2+ was low (e.g., 0.3 mM). Paired pacing exerted a small but significant inotropic effect in rat heart when extracellular Ca2+ was 2.5 mM, and a larger effect at lower Ca2+. As previously noted, hypertonic mannitol and paired pacing both produced significant positive effects in isolated cat heart at an extracellular Ca2+ concentration of 2.5 mM. D600 exerted less of a depressant effect on contractility in rat than in cat heart at concentrations of 10(-6)--10(-7) M. The data suggest that 1) in contrast to results in cat heart, the positive inotropic effect of hyperosmolality in isolated rat cardiac muscle is apparent only when extracellular calcium concentration is reduced; 2) the inotropic effect of paired pacing in rat heart is greatest at low Ca2+ levels, but persists to a lesser degree at extracellular calcium concentrations of 2.5 mM; and 3) D600-inhibitable calcium channels appear to be relatively less important in the maintenance of cardiac contractility in rat than in cat cardiac muscle.