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We read with interest the recent article by Agarwal and colleagues investigating the biologic significance of granulocyte colony stimulating factor (G-CSF) in stem-like subpopulations of neuroblastoma cells ([1][1]). While we find their observations provocative, and do not debate their demonstration

The transmembrane proto-oncogene receptor tyrosine kinase (RTK) ROS is an orphan receptor that is aberrantly expressed in neoplasms of the central nervous system. Here, we report the fusion of its carboxy-terminal kinase domain to the amino-terminal portion of a protein called FIG (Fused in Glioblastoma) in a human glioblastoma multiforme (GBM). By characterizing both FIG and ROS genes in normal and in U118MG GBM cells, we determined that an intra-chromosomal homozygous deletion of 240 kilobases on 6q21 is responsible for the formation of the FIG-ROS locus. The FIG-ROS transcript is encoded by 7 FIG exons and 9 ROS-derived exons. We also demonstrate that the FIG-ROS locus encodes for an in-frame fusion protein with a constitutively active kinase activity, suggesting that FIG-ROS may act as an oncogene. This is the first example of a fusion RTK protein that results from an intra-chromosomal deletion, and it represents the first fusion RTK protein isolated from a human astrocytoma.

Objectives. Data from the 1995–1996 and 1998–1999 Current Population Survey tobacco use supplements were used to examine smoking prevalence statistics by race/ethnicity and immigrant status. Methods. Smoking prevalence statistics were calculated, and these data were decomposed by country of birth for Asian immigrants to illustrate the heterogeneity in smoking rates present within racial/ethnic groups. Results. Except in the case of male Asian/Pacific Islanders, immigrants exhibited significantly lower smoking prevalence rates than nonimmigrants. However, rates varied according to country of birth. Conclusions. This research highlights the need to disaggregate health statistics by race/ethnicity, sex, immigrant status, and, among immigrants, country of birth. Data on immigrants’ health behaviors enhance the development of targeted and culturally sensitive public health smoking prevention programs.

Activating oncogenic mutations of receptor tyrosine kinases (RTKs) have been reported in several types of cancers. In many cases, genomic rearrangements lead to the fusion of unrelated genes to the DNA coding for the kinase domain of RTKs. All RTK-derived fusion proteins reported so far display oligomerization sequences within the 5′ fusion partners that are responsible for oncogenic activation. Here, we report a mechanism by which an altered RTK gains oncogenic potential in a glioblastoma cell line. A microdeletion on 6q21 results in the fusion of FIG , a gene coding for a Golgi apparatus-associated protein, to the kinase domain of the protooncogene c- ROS . The fused protein product FIG-ROS is a potent oncogene, and its transforming potential resides in its ability to interact with and become localized to the Golgi apparatus. Thus we have found a RTK fusion protein whose subcellular location leads to constitutive kinase activation and results in oncogenic transformation.

Housing needs is a concept of central importance to state and local planning in the United States (Landis and LeGates 2000). Roughly characterized as the number and type of housing units required to accommodate a population at a given standard of housing occupancy, the formulation of a quantified estimate of housing needs requires many assumptions that intertwine normative and empirical judgments. The overall aim of this article is to propose a needed theoretical framework and more rigorous methods for demographic component of housing needs estimates. Grounding this in the recent California experience helps to illustrate concepts with a concrete example. As demographic change continues to spread across the country, growing numbers of regions and cities can benefit from this study of the California experience. The article begins with a broad overview of the definition of housing needs, and then focuses on the central role of population growth and change in determining future construction needs. A pivotal issue is the instability over time of the empirical relationship between population and housing, as shown by comparison of household formation and homeownership rates from 1960 to 2000. A further issue is the sharp differences registered between different age groups, races, ethnicities, and nativity groups. Although disaggregation permits projections to capitalize on observed differences between groups, it also highlights the existence of inequities and the policy goal of reducing them.

This paper describes and compares the infant care practices and beliefs of Maori, Tongan, Samoan, Cook Islands, Niuean and Pakeha (European) caregivers residing in Auckland, New Zealand. Focusing on four areas--sources of support and advice; infant feeding; infant sleeping arrangements; and traditional practices and beliefs--it explores inter-ethnic similarities and differences and intra-ethnic tensions. The international literature indicates that there can be significant cultural variation in infant care practices and in the meanings attributed to them. There is, however, little New Zealand literature on this topic, despite its importance for effective health service and health message delivery. Participants were primary caregivers of infants under 12 months. An average of six focus groups were conducted within each ethnic group, resulting in a total of 37 groups comprising 150 participants. We found similarities across all ethnic groups in the perceived importance of breastfeeding and the difficulties experienced in establishing and maintaining this practice. The spectrum of behaviours ranged widely with differences most pronounced between Pacific caregivers, especially those Island-raised, and Pakeha caregivers, especially those in nuclear families. Amongst the former, norms included: the family as central in providing support and advice; infant bedsharing; abdominal rubbing during pregnancy; baby massage; and the importance of adhering to traditional protocols to ensure infant well-being. Amongst the latter, norms included: strong reliance on professional advice; looser family support networks; the infant sleeping in a cot; and adherence to Western biomedical understandings of health and illness. Maori caregivers bridged the spectrum created by these groups and exhibited a diverse range of practices. Intra-cultural differences were present in all groups indicating the dynamic nature of cultural practices. They were most evident between Pacific-raised and New Zealand-raised Pacific caregivers, with the latter attempting to marry traditional with Western beliefs and practices.

Aim. To evaluate the diagnostic utility of computed tomography (CT) obtained during prolonged febrile neutropenia in pediatric oncology patients. Methods. We evaluated the medical records of all patients with a malignant disease who had a CT examination during an episode of febrile neutropenia lasting for 4 days or more at Children's Hospital and Regional Medical Center in Seattle, WA, between January 1, 1997, and June 1, 1999. Results. CT was performed on 83 patients to evaluate 109 episodes of prolonged febrile neutropenia. Sixty-eight (62%) of the initial CT scans demonstrated abnormalities, leading to changes in therapy in 42 (39%). The diagnostic and therapeutic utility of CT varied by anatomic site. Abdominal and head/neck CT detected abnormalities in only 19 and 8% of studies, respectively, resulting in therapy changes in 9 and 4%, respectively. Sinus CT demonstrated abnormalities in 41% of cases and altered therapy in 24%. Chest CT had the highest diagnostic utility, with 49% of cases demonstrating abnormalities, leading to therapy alteration in 30%. CT was rarely abnormal in the absence of localizing signs or symptoms. In 55 instances 1 or more follow-up scans were done. Thirteen follow-up CT scans showed abnormalities that led to a change in therapy. Conclusions. CT-detected abnormalities frequently lead to alterations in therapy, particularly sinus and thoracic CT. Most patients with CT-detected abnormalities have symptoms or signs referable to the site of abnormality. Asymptomatic febrile neutropenic children rarely have CT findings that lead to a change in therapy.

In many databases, biocuration primarily involves literature curation, which usually involves retrieving relevant articles, extracting information that will translate into annotations and identifying new incoming literature. As the volume of biological literature increases, the use of text mining to assist in biocuration becomes increasingly relevant. A number of groups have developed tools for text mining from a computer science/linguistics perspective, and there are many initiatives to curate some aspect of biology from the literature. Some biocuration efforts already make use of a text mining tool, but there have not been many broad-based systematic efforts to study which aspects of a text mining tool contribute to its usefulness for a curation task. Here, we report on an effort to bring together text mining tool developers and database biocurators to test the utility and usability of tools. Six text mining systems presenting diverse biocuration tasks participated in a formal evaluation, and appropriate biocurators were recruited for testing. The performance results from this evaluation indicate that some of the systems were able to improve efficiency of curation by speeding up the curation task significantly (∼1.7- to 2.5-fold) over manual curation. In addition, some of the systems were able to improve annotation accuracy when compared with the performance on the manually curated set. In terms of inter-annotator agreement, the factors that contributed to significant differences for some of the systems included the expertise of the biocurator on the given curation task, the inherent difficulty of the curation and attention to annotation guidelines. After the task, annotators were asked to complete a survey to help identify strengths and weaknesses of the various systems. The analysis of this survey highlights how important task completion is to the biocurators’ overall experience of a system, regardless of the system’s high score on design, learnability and usability. In addition, strategies to refine the annotation guidelines and systems documentation, to adapt the tools to the needs and query types the end user might have and to evaluate performance in terms of efficiency, user interface, result export and traditional evaluation metrics have been analyzed during this task. This analysis will help to plan for a more intense study in BioCreative IV.

Calling the Station Home: Place and Identity in New Zealandis High Country. Michele D. Dominy. Lanham, MD: Rowman and Littlefield Publishers, 2001. xiii. 306 pp., figures, tables, maps, notes, bibliography, index.

Drugs directed toward oncoproteins have demonstrated efficacy while avoiding the systemic toxicity associated with standard chemotherapeutics. However, oncoproteins encoding for transcription factors, such as the MYC family, which are broadly implicated in many human cancers, are difficult to inhibit with small molecules or antibody based therapies. Thus, to target MYC-driven cancers, we have taken the approach of identifying druggable genes that exhibit a synthetic lethal relationship with aberrant MYC expression. Using an isogenic cell model system, we identified, via high throughput siRNA screening, more than 100 druggable genes that exhibit a synthetic lethal interaction with MYC (referred to as MYC-synthetic lethal genes, MYC-SL). Among the MYC-SL genes, we focused on casein kinase 1 epsilon (CSNK1e), whose relevance in MYC-driven human cancer was demonstrated by correlation between high levels of CSNK1e expression, MYCN amplification, and poor clinical prognosis in neuroblastoma cases. The requirement of CSNK1e for growth of neuroblastomas with MYCN amplification was validated in vivo by conditional knock-down and via a small molecule inhibitor of its activity. CSNK1e had previously been implicated in the regulation of WNT, however our data also suggest that in neuroblastoma the activity of CSNK1e is a key component of a positive autocrine circuit triggered by MYCN amplification that sustains both WNT and SHH pathways. In summary, our work elucidates a broad network of MYC synthetic lethal genes, perhaps a reflection of its widespread effects on gene expression, and demonstrates the power of high throughput siRNA screening as a rapid and unbiased approach to identify a network of synthetic lethal genes and potential new therapeutic targets functionally linked to a previously un-druggable oncogene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C135.

Annotation using Gene Ontology (GO) terms is one of the most important ways in which biological information about specific gene products can be expressed in a searchable, computable form that may be compared across genomes and organisms. Because literature-based GO annotations are often used to propagate functional predictions between related proteins, their accuracy is critically important. We present a strategy that employs a comparison of literature-based annotations with computational predictions to identify and prioritize genes whose annotations need review. Using this method, we show that comparison of manually assigned 'unknown' annotations in the Saccharomyces Genome Database (SGD) with InterPro-based predictions can identify annotations that need to be updated. A survey of literature-based annotations and computational predictions made by the Gene Ontology Annotation (GOA) project at the European Bioinformatics Institute (EBI) across several other databases shows that this comparison strategy could be used to maintain and improve the quality of GO annotations for other organisms besides yeast. The survey also shows that although GOA-assigned predictions are the most comprehensive source of functional information for many genomes, a large proportion of genes in a variety of different organisms entirely lack these predictions but do have manual annotations. This underscores the critical need for manually performed, literature-based curation to provide functional information about genes that are outside the scope of widely used computational methods. Thus, the combination of manual and computational methods is essential to provide the most accurate and complete functional annotation of a genome. Database URL: http://www.yeastgenome.org.

A comparison of folk categories of types of drinker and recorded alcohol consumption data, both derived from New Zealand women, with established risky levels of drinking indicates that there is some correspondence between folk categories of non-‘ordinary’ drinking and drinking at epidemiologically defined risky levels. However, of more importance to the majority of women drinkers, women's drinking which is completely socially acceptable and certainly regarded as quite ‘ordinary’, can also be at or well above risky levels. These findings are discussed in the context of the increasing normalisation of alcohol in women's lives. I argue that they support a ‘reduced consumption’ rather than a ‘safe limits’ alcohol policy.

DNA transposons such as Sleeping Beauty (SB) have been shown to be effective vectors for gene therapy. However, one limitation of the SB system is its regulation by a phenomenon known as overproduction inhibition (OPI), in which elevated concentrations of transposase enzyme inhibit the transposition reaction. In an attempt to characterize the mechanism behind this process, we studied the structure/function relationship of the wild-type SB10 protein|[mdash]|focusing on the region linking the N-terminal DNA binding domain and the C-terminal catalytic domain. This area of SB10 is not only highly conserved (55-60%) among the Tc1/Mariner family of transposases, but has also been previously implicated in adversely affecting SB's DNA binding ability. We first tested whether regions downstream of the transposase N-terminus contribute to SB10 DNA binding activity by analyzing various SB deletion mutants in a yeast one-hybrid assay for transposase-transposon interactions. Results show that the ability of the protein to bind transposon DNA increases exponentially in a manner that is proportional with the size of the C-terminal deletion. Mutational analyses of this inter-domain linker region indicate that it is important for transposase function since nearly all mutants analyzed were either completely inactive or 2-3 fold hyperactive in a quantitative transposition assay. To ascertain whether this newly-identified functional domain contributes to OPI, SB10 and five hyperactive mutants were assayed for transposition activity at increasing transposase:transposon ratios. All mutants demonstrated altered OPI titration curves as compared to SB10, with two distinct mutant classes emerging. Class 1 mutants yielded a curve which was shifted towards higher transposase:transposon ratios, but had the same maximum activity as the SB10 curve. In contrast, Class 2 mutants had transposition levels only reaching |[sim]|50% that of SB10. However, these curves were also shifted toward higher amounts of transposase. Based on the similar shift in curves for both types of mutants, we concluded that mutations in the linker domain resulted in an increased OPI threshold. To elucidate the molecular mechanism behind this change in SB regulation, we tested the binding abilities of these linker domain mutants using our yeast one-hybrid assay. We found that all the inactive transposase mutants were able to bind transposon DNA at least as well as SB10 and some of hyperactive mutants exhibited up to a 4-fold change in DNA binding ability. Though some of the mutants did show a dramatic change in transposon binding, we observed no obvious overall correlation between binding and transpositional regulation. However, based on an altered OPI curve of an N-terminal transposase mutant with known increased affinity for the transposon, we suspect that some relationship between binding and regulation exists. Although we do not as yet completely understand the mechanism behind OPI, our results indicate that it is possible to remove some of the stringency of SB self-inhibition. Through continued study of this regulatory process, we hope to eventually overcome this obstacle and improve the efficiency and utility of SB as a vehicle for gene transfer.

Top of pageAbstract Vectors from adeno-associated virus (AAV) appear very promising for use in some gene therapy applications, and have even shown success in animal models. Different serotypes of AAV have been found, each having different properties. For example, it has been shown that AAV-8 uncoats more readily than AAV-2 in the nuclei of hepatocytes in mouse liver, possibly explaining the more robust AAV-8 |[ndash]|transduction efficiencies (Thomas et al., Journal of Virology, 2004, 78 (6) p. 3110|[ndash]|3224). Our goal is to better understand the mechanisms of AAV capsid uncoating in vivo. One approach is to identify cellular proteins that interact with the capsid, and perhaps establish if certain proteins interact with the capsids in a serotype-specific manner. Towards this end, we used a yeast two-hybrid screen to identify cellular proteins that interact with different domains of the AAV-8 capsid. In one of the screens, using the middle portion of the AAV capsid protein VP3 as bait against a mouse liver cDNA library, we found that over half of the library clones recovered encoded part of the same hypothetical ORF AK014478. These clones did interact with the corresponding portion of the AAV-2 capsid, indicating this interaction is not serotype-specific. However, they did not interact with the C- or N-terminal portions of the capsid protein. Homologues for this hypothetical protein have been found only in certain vertebrates and insects, and the human homologue has been shown to localize to the mitochondria. Additional clones isolated from these and related screens are currently being identified, and their association with AAV-capsid proteins are being verified by independent assays. By identifying these cellular proteins such as the one encoded by AK014478 we can better understand the mechanisms behind AAV entry and uncoating in the cell, and design better vectors for gene transfer.

The Sleeping Beauty (SB) transposon system mediates stable genomic integration by a cut-and-paste transposition process and has shown tremendous success as a therapeutic vehicle for in vivo gene delivery. Nevertheless, the overall rate of transposition with SB is still insufficient for many clinical applications. Herein, we generated 141 missense mutants for the SB transposase and screened each for altered gene transfer activity in human cells using a genetic assay. We show that despite equivalent steady-state levels of wild-type (WT) and mutant transposases in transfected cells, many mutations introduced into the N-terminal DNA-binding domain (n=10) and C-terminal catalytic core domain (n=6) resulted in 2- to 4-fold higher transpositional efficiencies compared to WT. In order to better understand the transposition reaction for potential future development, we compared the functional activities of these hyperactive recombinases to that of the WT transposase using a combination of PCR and EMSAs. Results of these studies identified a number of pathways that contribute to improved transposase activity, including an altered affinity of the transposase for transposon ends, transposase conformational changes, enhanced donor cleavage activity, and increased rates of strand transfer. Importantly, many of these hyperactive SB (HSB) mutations functioned synergistically and, when combined with a hyperactive transposon, elevated transposition by 14-fold compared to the first-generation transposon system. Moreover, although WT and mutant transposases were negatively regulated at high enzyme concentrations, the HSB variants supported ~10-fold higher transpositional activity at all experimental doses, suggesting that lower doses could be administered to further improve the safety profile for SB in vivo. In addition, the use of HSB instead of the WT transposase dramatically improved the integration frequency of larger-sized elements, including ones greater than 14 kb in length. These HSB mutants should greatly extend the carrying capacity for SB, thus overcoming one of this system's major limitations. Finally, we studied the long-term in vivo persistence of two transposon reporters, β-galactosidase and human coagulation factor IX (hFIX), in the livers of adult mice following a single administration of WT- or hyperactive transposase-expressing plasmids. Results of these analyses demonstrated that these mutated enzymes could also greatly enhance SB's gene transfer capabilities in vivo, supporting stable integration in an estimated 57% of transfected mouse hepatocytes, compared to ~8% for the WT. This level of transposition was sufficient to maintain ~7-fold higher serum hFIX levels (955 ng/ml ± 241 ng/ml) than achieved in SB-expressing animals (140 ng/ml ± 56 ng/ml) for a period of 4 months (length of study). This level of hFIX obtained with the HSB mutant is ~19% of normal human levels and is well within a curative range capable of converting a severely affected hemophilia B patient to one with a much milder phenotype. Therefore, our work demonstrates the potential for improved in vivo gene delivery through directed transposase evolution, and provides a number of important insights into Sleeping Beauty transposon biology that could be exploited for future development.

Top of pageAbstract The Sleeping Beauty (SB) transposon system has been shown as an effective vector to mediate stable, long-term gene transfer. We have previously identified SB hyperactive mutants through an alanine scan of the DNA binding region of the transposase. In total, 96 single-substitution mutants were generated and those individual missense mutations leading to increased activity were combined to generate four hyperactive Sleeping Beauty (HSB) mutants. In this study, we have begun to delve into the mechanism behind the increased activity of these mutants by comparing the four HSB mutants and wild-type transposase for their ability to bind the DNA-recognition sites. Truncated (N123) derivatives of wild-type and hyperactive SB proteins were generated by in vitro transcription/translation and tested for their ability to bind to oligos corresponding to transposase binding sites in an electrophoretic mobility shift assay. Each SB transposon inverted repeat contains two transposase binding sites, an outer binding site (OBS) adjacent to the transposon boundary and an inner binding site (IBS) internally situated about 165bp from the OBS. We found that there was no change in the binding affinities of the hyperactive mutants versus wild-type for the IBS but that binding to the OBS was increased 2–4 fold. Our results in conjunction with previous work, where it was shown that simply increasing the binding affinity of the transposase at the transposon terminal end causes the abolishment of transposition (Cui et al, J Mol Biol, 1221–1235; 2002), suggest that the mechanisms underlying hyperactivity may be due to an enhanced ability of these mutants to form an active synaptic complex and we are in the process of investigating this possibility. We are also examining all of the single-substitution mutants to determine which individual residues contribute to increased binding affinity. Through these experiments we hope to gain additional insight into the mechanisms involved in transposase function which, in turn, will lead to further enhancement of SB as a tool for gene transfer.

McCarthy and his colleagues raise interesting questions about smoking behavior in other countries. Our research, however, is restricted to the smoking behavior of foreign-born men and women in the United States. We apologize for any confusion about the meaning of “country of origin.” We follow the convention of most immigration research by using country of birth. That should resolve the doubts expressed by the letter writers regarding the treatment of migrants who stop over in, for example, Canada before settling in the United States. We regret that the tobacco use supplement data from the Current Population Survey prevent us from exploring the use of alternative tobacco products such as bidis or paan. The data in question pertain primarily to cigarette smoking. There may well be cultural differences in what is considered “smoking.” This is always a challenge when making cross-cultural comparisons.

Sequencing and annotation of the entire Saccharomyces cerevisiae genome has made it pos- sible to gain a genome-wide perspective on yeast genes and gene products. To make this information available on an ongoing basis, the Saccharomyces 20Genome Database (SGD) (http://www.yeastgenome. org/) has created the Genome Snapshot (http://db. yeastgenome.org/cgi-bin/genomeSnapShot.pl). The Genome Snapshot summarizes the current state of knowledge about the genes and chromosomal fea- 25tures of S.cerevisiae. The information is organized into two categories: (i) number of each type of chro- mosomal feature annotated in the genome and (ii) number and distribution of genes annotated to Gene Ontology terms. Detailed lists are accessible 30through SGD's Advanced Search tool (http://db. yeastgenome.org/cgi-bin/search/featureSearch), and all the data presented on this page are available from the SGD ftp site (ftp://ftp.yeastgenome.org/ yeast/).

Background: Learning the function of genes is a major goal of computational genomics. Methods for inferring gene function have typically fallen into two categories: 'guilt-by-profiling', which exploits correlation between function and other gene characteristics; and 'guilt-by-association', which transfers function from one gene to another via biological relationships. Results: We have developed a strategy ('Funckenstein') that performs guilt-by-profiling and guilt-by-association and combines the results. Using a benchmark set of functional categories and input data for protein-coding genes in Saccharomyces cerevisiae, Funckenstein was compared with a previous combined strategy. Subsequently, we applied Funckenstein to 2,455 Gene Ontology terms. In the process, we developed 2,455 guilt-by-profiling classifiers based on 8,848 gene characteristics and 12 functional linkage graphs based on 23 biological relationships. Conclusion: Funckenstein outperforms a previous combined strategy using a common benchmark dataset. The combination of 'guilt-by-profiling' and 'guilt-by-association' gave significant improvement over the component classifiers, showing the greatest synergy for the most specific functions. Performance was evaluated by cross-validation and by literature examination of the top-scoring novel predictions. These quantitative predictions should help prioritize experimental study of yeast gene functions.