bluuebunny/crossref_metadata_2025 huggingface dataset:
Low striatal serotonin transporter protein in a human polydrug MDMA (ecstasy) user: a case study
Authors: Kish SJ , Fitzmaurice PS , Chang LJ , Furukawa Y. , Tong J.
Published: October 2008

Evidence that the widely used methamphetamine analog MDMA (3,4-methylenedioxymethamphetamine, ecstasy) might damage brain serotonin neurones in humans is derived from imaging investigations showing variably decreased binding of radioligands to the serotonin transporter (SERT), a marker of serotonin neurones. However, in humans, it is not known whether low SERT binding reflects actual loss of SERT protein itself. As this question can only be answered in post-mortem brain, we measured protein levels of SERT and that of the rate-limiting serotonin-synthesizing enzyme tryptophan hydroxylase (TPH) in autopsied brain of a high-dose MDMA user. As compared with control values, SERT protein levels were markedly (−48% to −58%) reduced in striatum (caudate, putamen) and occipital cortex and less affected (−25%) in frontal and temporal cortices, whereas TPH protein was severely decreased in caudate and putamen (−68% and −95%, respectively). The magnitude of the striatal SERT protein reduction was greater than the SERT binding decrease typically reported in imaging studies. Although acknowledging limitations of a case study, these findings extend imaging data based on SERT binding and suggest that high-dose MDMA exposure could cause loss of two key protein markers of brain serotonin neurones, a finding compatible with either physical damage to serotonin neurones or downregulation of components therein.
The Environment, Energy, and the Tinbergen Rule
Authors: Knudson William A.
Published: October 2008

Higher energy prices and the growing concern about global warming have led to a number of policy goals and targets designed to curb global warming and/or the development of alternative sources of energy. However, the Tinbergen Rule states that for each and every policy target there must be at least one policy tool. If there are fewer tools than targets, then some policy goals will not be achieved. Further complicating the public policy environment are the facts that some policy tools affect more than one target, some tools help achieve more than one target, and others, while meeting one target, make meeting other targets more difficult. Also, some targets are more efficient than others. If policy makers are going to be able to effectively meet their environmental and energy goals, a series of policy tools need to be developed.
Long-Term Effects of Bupivacaine on Cartilage in a Rabbit Shoulder Model
Authors: Gomoll Andreas H. , Yanke Adam B. , Kang Richard W. , Chubinskaya Susan , Williams James M. , Bach Bernard R. , Cole Brian J.
Published: October 2008

Background

Previous investigations have reported on the chondrotoxicity of bupivacaine in short-term in vivo and in vitro models. This study was designed to provide additional information on the long-term effects of bupivacaine infusion on articular cartilage in an established rabbit shoulder model.

Hypothesis

Infusion of bupivacaine into the rabbit shoulder will have long-term deleterious effects on articular cartilage.

Study Design

Controlled laboratory study.

Methods

Thirty-six rabbits were randomized into 3 groups and were infused over 48 hours with saline (S), bupivacaine alone (B), or bupivacaine with epinephrine (B+E) into the glenohumeral joint. Animals were sacrificed after 3 months, and tissue samples were analyzed with live/dead cell assay, proteoglycan (PG) synthesis and content assays, and conventional histological evaluation.

Results

No macroscopic or radiographic changes were detected in the infused shoulders. Sulfate uptake of infused shoulders relative to controls was elevated to 112% ± 39% (S), 166% ± 67% (B), and 210% ± 127% (B+E). Statistical analysis of PG content demonstrated significantly increased levels in bupivacaine groups compared with saline. There were no significant differences among groups in cell count, percentage of living cells, or histological grade.

Conclusions

No permanent impairment of cartilage function was detected 3 months after intra-articular infusion of bupivacaine. Cartilage metabolism was increased, indicating a possible reparative response. This suggests that, at least in the model used, articular cartilage has the ability to recover from the chondrotoxic effects of bupivacaine infusion. Before extrapolating these results to human cartilage, other factors including underlying cartilage injury or disease, decreased chondrocyte density, and increased bupivacaine dosing need to be taken into account.

Clinical Relevance

Bupivacaine toxicity has recently been implicated in the development of chondrolysis after arthroscopic shoulder procedures, but these findings suggest that additional noxious stimuli might be required before permanent damage ensues.
Introduction: intimacy in research
Authors: Fraser Mariam , Puwar Nirmal
Published: October 2008

The introduction to this special issue addresses the production of intimacy in the labour of research. It explores the sensory, emotional and affective relations which form an integral, if often invisible, part of the process through which researchers engage with, produce, understand and translate `research'. The article argues that these processes inform the making of knowledge, shape power relations and enable or constrain the practical negotiation of ethical problems. These issues are not, however, often foregrounded in debates on methods or methodology and are frequently erased from researchers' own accounts of their work. The article explores some of the possible reasons for this, which include institutional and cultural conventions of academic practice, the historical legacies with which disciplines often struggle, and the difficult issues and decisions that individual researchers face as they try to negotiate the relations between scholarly research and personal relationships across time, and between scholarly research and, for example, creativity, fiction, or sensationalism. The article concludes with a review of the main themes in the special issue, focusing in particular on the ways in which the contributors use the concept of intimacy to challenge the boundaries between creativity and analysis; spatial and temporal proximity and distance; freedom and censorship; subjects and objects.
Inventing Iris: negotiating the unexpected spatialities of intimacy
Authors: Parry Bronwyn C.
Published: October 2008

This article explores a number of questions about the relationship between intimacy and research that were bought into sharp focus for me by a disturbing event: my unexpected encounter with Iris Murdoch's archived brain. In considering how very intimate experiences such as these are both constructed and narrated to wider audiences, I begin by exploring the nature of intimacy itself. Here I argue that intimacy is the product of not only social but spatial relations, relations that may, in contrast to popular conceptions, be `stretched out' to create what I call here `distributed spaces of intimacy'. In exploring the role that material artefacts can play as objects that create essential points of interface between individuals and communities that are geographically and socially distant, I also draw attention to the necessarily partial and relational nature of intimacy. In the final section of the article I turn to consider the impact that intensely personal experiences may have on research methodologies and the vexatious question of how, if at all, it is possible to speak of, or report them, without transgressing important social, moral and ethical conventions.
A method of intuition: becoming, relationality, ethics
Authors: Coleman Rebecca
Published: October 2008

This article examines social research on the relations between (young) women's bodies and images through Bergson's method of intuition, which suggests that the only way a thing can be known is through coinciding with the uniqueness of its becoming. I suggest that in this aim, intuition is, necessarily, an intimate research method. Rather than apply Bergson's argument to this area of social research, I examine the resonances between his philosophical method and the moves within social research to attend to the performativity, creativity or inventiveness of research methods. With a focus on my own research, which explored the relations between 13 girls' bodies and images from a feminist-Deleuzian position, I argue here that the interconnected issues of becoming, uniqueness and coincidence that Bergson raises connect with concerns in social research about ontology, concepts and methods. In particular, I suggest that relationality is crucial to these connections. Drawing through the significance of relations, I argue that intimate, intuitive research is desirable because of the ethics that it opens up and enables; ethics intimate in attention to the becoming unique to the object at stake in research and in the attempt to coincide with this uniqueness.
Violence Against Women
Authors: Oweis Arwa , Gharaibeh Muntaha , Al-Natour Ahlam , Froelicher Erika
Published: October 2008

The descriptive qualitative study documents experiences of Jordanian women abused by their husbands. Thirteen married women living with an abusive husband were recruited from a major primary healthcare center. The women described forms, intensity, and consequences of their husband's acts. Despite their anguish, women justified, normalized, and tolerated abuse; this acceptance reflected their cultural values and norms. Implications for nursing practice include (a) identifying abused women, (b) empowering them with knowledge to change their sociocultural perceptions of marital relationships, (c) designing interventions that address their needs, and (d) educating health care providers to take a role in addressing spousal abuse.
Telling Interviewers About Sexual Abuse
Authors: Lippert Tonya , Cross Theodore P. , Jones Lisa , Walsh Wendy
Published: October 2008

This study aims to identify characteristics that predict full disclosure by victims of sexual abuse during a forensic interview. Data came from agency files for 987 cases of sexual abuse between December 2001 and December 2003 from Children's Advocacy Centers (CACs) and comparison communities within four U.S. states. Cases of children fully disclosing abuse when interviewed were compared to cases of children believed to be victims who gave no or partial disclosures. The likelihood of disclosure increased when victims were girls, a primary caregiver was supportive, and a child's disclosure instigated the investigation. The likelihood of disclosure was higher for children who were older at abuse onset and at forensic interview (each age variable having an independent effect). Communities differed on disclosure rate, with no difference associated with having a CAC. Findings suggest factors deserving consideration prior to a forensic interview, including organizational and community factors affecting disclosure rates.
Cognitive Deficits and Reduced Insight in Primary Progressive Aphasia
Authors: Banks Sarah Jane , Weintraub Sandra
Published: October 2008

Primary progressive aphasia (PPA) is a form of dementia caused by frontotemporal lobar degeneration. Unlike aphasia due to stroke, in which the association between particular aphasia profiles and insight has been well characterized, this relationship has not been investigated in PPA. Reduced insight is seen in other neurological conditions, but tends to involve right hemisphere damage, whereas PPA is predominantly a left hemisphere disorder. The aim of the current study was to examine whether fluent aphasia with less meaningful speech output, associated with diminished insight in stroke, is also characteristic of PPA patients with reduced insight. Fourteen PPA patients were studied. Results indicated that reduced information content in speech and poor performance on a nonlanguage test, the Pyramids and Palm Trees test, predicted reduced insight. This study has implications for the anatomical network involved in insight and clinical implications in terms of selecting interventions appropriate for individual patients with PPA.
The Evolution of John Mezirow's Transformative Learning Theory
Authors: Kitchenham Andrew
Published: October 2008

This article is a review of Mezirow's transformative learning from its inception to the latest definition. The review builds on Taylor's earlier discussions, but unlike his review, this history of transformative learning relies predominantly on Mezirow's publications to authenticate the discussion with support from the extant literature. The article begins with Mezirow's explanation of the stages of transformative learning, continues with the influences on the theory, transitions into the criticisms, and concludes with a discussion of its evolution and development.
Metatheoretical Prospects for the Field of Transformative Learning
Authors: Gunnlaugson Olen
Published: October 2008

This article examines the complex mosaic of first- and second-wave theories of transformative learning and recommends the development of a subfield of meta-inquiry. Central to this undertaking is the need for metatheorizing, to which this article outlines a rationale with specific criteria for developing a metatheoretical discourse and more comprehensive metatheoretical frameworks. It then goes on to examine the strengths and limitations of Wilber's AQAL model as a possible candidate for working with a meta-analysis of first- and second-wave contributions within the field.
Functional alteration of red blood cells by a megadalton protein of Plasmodium falciparum
Authors: Glenister Fiona K. , Fernandez Kate M. , Kats Lev M. , Hanssen Eric , Mohandas Narla , Coppel Ross L. , Cooke Brian M.
Published: October 2008

Proteins exported from Plasmodium falciparum parasites into red blood cells (RBCs) interact with the membrane skeleton and contribute to the pathogenesis of malaria. Specifically, exported proteins increase RBC membrane rigidity, decrease deformability, and increase adhesiveness, culminating in intravascular sequestration of infected RBCs (iRBCs). Pf332 is the largest (>1 MDa) known malaria protein exported to the RBC membrane, but its function has not previously been determined. To determine the role of Pf332 in iRBCs, we have engineered and analyzed transgenic parasites with Pf332 either deleted or truncated. Compared with RBCs infected with wild-type parasites, mutants lacking Pf332 were more rigid, were significantly less adhesive to CD36, and showed decreased expression of the major cytoadherence ligand, PfEMP1, on the iRBC surface. These abnormalities were associated with dramatic morphologic changes in Maurer clefts (MCs), which are membrane structures that transport malaria proteins to the RBC membrane. In contrast, RBCs infected with parasites expressing truncated forms of Pf332, although still hyperrigid, showed a normal adhesion profile and morphologically normal MCs. Our results suggest that Pf332 both modulates the level of increased RBC rigidity induced by P falciparum and plays a significant role in adhesion by assisting transport of PfEMP1 to the iRBC surface.
Aberrant DNA methylation is a dominant mechanism in MDS progression to AML
Authors: Jiang Ying , Dunbar Andrew , Gondek Lukasz P. , Mohan Sanjay , Rataul Manjot , O'Keefe Christine , Sekeres Mikkael , Saunthararajah Yogen , Maciejewski Jaroslaw P.
Published: October 2008

Myelodysplastic syndromes (MDSs) are clonal hematologic disorders that frequently represent an intermediate disease stage before progression to acute myeloid leukemia (AML). As such, study of MDS/AML can provide insight into the mechanisms of neoplastic evolution. In 184 patients with MDS and AML, DNA methylation microarray and high-density single nucleotide polymorphism array (SNP-A) karyotyping were used to assess the relative contributions of aberrant DNA methylation and chromosomal deletions to tumor-suppressor gene (TSG) silencing during disease progression. Aberrant methylation was seen in every sample, on average affecting 91 of 1505 CpG loci in early MDS and 179 of 1505 loci after blast transformation (refractory anemia with excess blasts [RAEB]/AML). In contrast, chromosome aberrations were seen in 79% of early MDS samples and 90% of RAEB/AML samples, and were not as widely distributed over the genome. Analysis of the most frequently aberrantly methylated genes identified FZD9 as a candidate TSG on chromosome 7. In patients with chromosome deletion at the FZD9 locus, aberrant methylation of the remaining allele was associated with the poorest clinical outcome. These results indicate that aberrant methylation can cooperate with chromosome deletions to silence TSG. However, the ubiquity, extent, and correlation with disease progression suggest that aberrant DNA methylation is the dominant mechanism for TSG silencing and clonal variation in MDS evolution to AML.
Wnt3a deficiency irreversibly impairs hematopoietic stem cell self-renewal and leads to defects in progenitor cell differentiation
Authors: Luis Tiago C. , Weerkamp Floor , Naber Brigitta A. E. , Baert Miranda R. M. , de Haas Edwin F. E. , Nikolic Tatjana , Heuvelmans Sjanneke , De Krijger Ronald R. , van Dongen Jacques J. M. , Staal Frank J. T.
Published: October 2008

Canonical Wnt signaling has been implicated in various aspects of hematopoiesis. Its role is controversial due to different outcomes between various inducible Wnt-signaling loss-of-function models and also compared with gain-of-function systems. We therefore studied a mouse deficient for a Wnt gene that seemed to play a nonredundant role in hematopoiesis. Mice lacking Wnt3a die prenatally around embryonic day (E) 12.5, allowing fetal hematopoiesis to be studied using in vitro assays and transplantation into irradiated recipient mice. Here we show that Wnt3a deficiency leads to a reduction in the numbers of hematopoietic stem cells (HSCs) and progenitor cells in the fetal liver (FL) and to severely reduced reconstitution capacity as measured in secondary transplantation assays. This deficiency is irreversible and cannot be restored by transplantation into Wnt3a competent mice. The impaired long-term repopulation capacity of Wnt3a−/− HSCs could not be explained by altered cell cycle or survival of primitive progenitors. Moreover, Wnt3a deficiency affected myeloid but not B-lymphoid development at the progenitor level, and affected immature thymocyte differentiation. Our results show that Wnt3a signaling not only provides proliferative stimuli, such as for immature thymocytes, but also regulates cell fate decisions of HSC during hematopoiesis.
NKT-cell help to B lymphocytes can occur independently of cognate interaction
Authors: Tonti Elena , Galli Grazia , Malzone Carmine , Abrignani Sergio , Casorati Giulia , Dellabona Paolo
Published: October 2008

CD4+ T (Th)–cell help to B lymphocytes requires cognate interaction and CD40 engagement. Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that recognize αgalactosylceramide (αGalCer) presented by CD1d, and can help B-cell responses. We asked whether αGalCer-activated iNKT cells help B lymphocytes through cognate interaction, or indirectly, via enhancement of Th-B–cell interaction. After immunization with protein Ags and αGalCer, antibody titers were assessed in wild-type or splenectomized mice, and in bone marrow radiation chimeras lacking CD1d or CD40 expression on B lymphocytes, or expressing CD1d or MHC II disjointly on antigen-presenting cells (APCs). We find that αGalCer-dependent enhancement of B-cell response (1) can occur when B cells do not express CD1d but express CD40; (2) requires that iNKT and Th cells interact with the same APCs that coexpress both CD1d and MHC-II; and (3) takes place without spleen. These findings demonstrate αGalCer-induced help for antibody responses can occur without cognate iNKT/B-cell interaction, and suggest this help entails activation of APCs by iNKT cells, which in turn activate Th cells and their helper functions for B cells. Thus, the αGalCer-induced help recapitulates the function of classical adjuvants that stimulate the innate immune system to support adaptive immune responses.
Host CD4+CD25+ T cells can expand and comprise a major component of the Treg compartment after experimental HCT
Authors: Bayer Allison L. , Jones Monica , Chirinos Jackeline , de Armas Lesley , Schreiber Taylor H. , Malek Thomas R. , Levy Robert B.
Published: October 2008

Reconstitution of the recipient lymphoid compartment following hematopoietic cell transplantation (HCT) is typically delayed. The present studies investigated the residual host CD4+CD25+Foxp3+ (Treg) compartment after several conditioning regimens, including T cell–depleted and T cell–replete HCT and observed (1) a small number of recipient Treg cells survived aggressive conditioning; (2) the surviving, that is, residual Tregs underwent marked expansion; and (3) recipient CD4+FoxP3+ cells composed the majority of the Treg compartment for several months post-syngeneic HCT. Notably, residual Tregs also dominated the compartment post-HCT with T cell–depleted (TCD) major histocompatibility complex–matched allogeneic bone marrow but not following T cell–replete transplantations. The residual Treg cell compartment was functionally competent as assessed by in vitro lymphoid suppression and in vivo autoimmune disease transfer assay. These observations support the notion that functional host Tregs initially occupy a niche in lymphopenic transplantation recipients, undergo significant expansion, and contribute to the compartment for an extended period before donor-derived CD4+FoxP3+ T cells eventually compose the majority of the compartment. In total, the findings suggest that the presence of host Tregs may be important to consider regarding elicitation of immune (eg, antitumor, vaccine) responses in recipients during the early post-transplant period involving autologous and certain allogeneic HCT regimens.
Methylation screening of the TGFBI promoter in human lung and prostate cancer by methylation-specific PCR
Authors: Shah Jinesh N , Shao Genze , Hei Tom K , Zhao Yongliang
Published: October 2008

Background

Hypermethylation of the TGFBI promoter has been shown to correlate with decreased expression of this gene in human tumor cell lines. In this study, we optimized a methylation-specific polymerase chain reaction (MSP) method and investigated the methylation status of the TGFBI promoter in human lung and prostate cancer specimens.

Methods

Methylation-specific primers were designed based on the methylation profiles of the TGFBI promoter in human tumor cell lines, and MSP conditions were optimized for accurate and efficient amplification. Genomic DNA was isolated from lung tumors and prostatectomy tissues of prostate cancer patients, bisulfite-converted, and analyzed by MSP.

Results

Among 50 lung cancer samples, 44.0% (22/50) harbored methylated CpG sites in the TGFBI promoter. An analysis correlating gene methylation status with clinicopathological cancer features revealed that dense methylation of the TGFBI promoter was associated with a metastatic phenotype, with 42.9% (6/14) of metastatic lung cancer samples demonstrating dense methylation vs. only 5.6% (2/36) of primary lung cancer samples (p < 0.05). Similar to these lung cancer results, 82.0% (41/50) of prostate cancer samples harbored methylated CpG sites in the TGFBI promoter, and dense methylation of the promoter was present in 38.9% (7/18) of prostate cancer samples with the feature of locoregional invasiveness vs. only 19.4% (6/31) of prostate cancer samples without locoregional invasiveness (p < 0.05). Furthermore, promoter hypermethylation correlated with highly reduced expression of the TGFBI gene in human lung and prostate tumor cell lines.

Conclusion

We successfully optimized a MSP method for the precise and efficient screening of TGFBI promoter methylation status. Dense methylation of the TGFBI promoter correlated with the extent of TGFBI gene silencing in tumor cell lines and was related to invasiveness of prostate tumors and metastatic status of lung cancer tumors. Thus, TGFBI promoter methylation can be used as a potential prognostic marker for invasiveness and metastasis in prostate and lung cancer patients, respectively.
Performance of mitochondrial DNA mutations detecting early stage cancer
Authors: Jakupciak John P , Maragh Samantha , Markowitz Maura E , Greenberg Alissa K , Hoque Mohammad O , Maitra Anirban , Barker Peter E , Wagner Paul D , Rom William N , Srivastava Sudhir , Sidransky David , O'Connell Catherine D
Published: October 2008

Background

Mutations in the mitochondrial genome (mtgenome) have been associated with cancer and many other disorders. These mutations can be point mutations or deletions, or admixtures (heteroplasmy). The detection of mtDNA mutations in body fluids using resequencing microarrays, which are more sensitive than other sequencing methods, could provide a strategy to measure mutation loads in remote anatomical sites.

Methods

We determined the mtDNA mutation load in the entire mitochondrial genome of 26 individuals with different early stage cancers (lung, bladder, kidney) and 12 heavy smokers without cancer. MtDNA was sequenced from three matched specimens (blood, tumor and body fluid) from each cancer patient and two matched specimens (blood and sputum) from smokers without cancer. The inherited wildtype sequence in the blood was compared to the sequences present in the tumor and body fluid, detected using the Affymetrix Genechip® Human Mitochondrial Resequencing Array 1.0 and supplemented by capillary sequencing for noncoding region.

Results

Using this high-throughput method, 75% of the tumors were found to contain mtDNA mutations, higher than in our previous studies, and 36% of the body fluids from these cancer patients contained mtDNA mutations. Most of the mutations detected were heteroplasmic. A statistically significantly higher heteroplasmy rate occurred in tumor specimens when compared to both body fluid of cancer patients and sputum of controls, and in patient blood compared to blood of controls. Only 2 of the 12 sputum specimens from heavy smokers without cancer (17%) contained mtDNA mutations. Although patient mutations were spread throughout the mtDNA genome in the lung, bladder and kidney series, a statistically significant elevation of tRNA and ND complex mutations was detected in tumors.

Conclusion

Our findings indicate comprehensive mtDNA resequencing can be a high-throughput tool for detecting mutations in clinical samples with potential applications for cancer detection, but it is unclear the biological relevance of these detected mitochondrial mutations. Whether the detection of tumor-specific mtDNA mutations in body fluidsy this method will be useful for diagnosis and monitoring applications requires further investigation.
Circulating chromogranin A reveals extra-articular involvement in patients with rheumatoid arthritis and curbs TNF-α-elicited endothelial activation
Authors: Di Comite Gabriele , Rossi Carlo M , Marinosci Alessandro , Lolmede Karine , Baldissera Elena , Aiello Patrizia , Mueller Ruediger B , Herrmann Martin , Voll Reinhard E , Rovere-Querini Patrizia , Sabbadini Maria Grazia , Corti Angelo , Manfredi Angelo A
Published: October 2008

TNF-α plays an important role in the natural history of rheumatoid arthritis (RA), a systemic disease characterized by endothelial activation and synovial involvement with bone erosions. Neuroendocrine signals contribute as well to RA, but their role is poorly understood. We measured in 104 RA patients and in an equal number of sex- and age-matched, healthy controls the blood levels of chromogranin A (CgA), a candidate marker linking the neuroendocrine system to TNF-α-mediated vascular inflammation. CgA levels were significantly higher in patients with RA and remained stable over time. High levels of CgA were significantly associated with severe extra-articular manifestations, namely pulmonary fibrosis, rheumatoid vasculitis, serositis, and peripheral neuropathy. RA sera curbed the response of human microvascular endothelial cells to TNF-α, as assessed by the expression of ICAM-1, the release of MCP-1/CCL2, and the export of nuclear high-mobility group box 1; the effect abated in the presence of anti-CgA antibodies. The efficacy of the blockade was significantly correlated with the CgA concentration in the serum. The recombinant aminoterminal portion of CgA, corresponding to residues 1–78, had similar inhibitory effects on endothelial cells challenged with TNF-α. Our results suggest that enhanced levels of CgA identify patients with extra-articular involvement and reveal a negative feedback loop that limits the activation of endothelial cells in RA.
An FGF autocrine loop initiated in second heart field mesoderm regulates morphogenesis at the arterial pole of the heart
Authors: Park Eon Joo , Watanabe Yusuke , Smyth Graham , Miyagawa-Tomita Sachiko , Meyers Erik , Klingensmith John , Camenisch Todd , Buckingham Margaret , Moon Anne M.
Published: October 2008

In order to understand how secreted signals regulate complex morphogenetic events, it is crucial to identify their cellular targets. By conditional inactivation of Fgfr1 and Fgfr2 and overexpression of the FGF antagonist sprouty 2 in different cell types, we have dissected the role of FGF signaling during heart outflow tract development in mouse. Contrary to expectation, cardiac neural crest and endothelial cells are not primary paracrine targets. FGF signaling within second heart field mesoderm is required for remodeling of the outflow tract: when disrupted, outflow myocardium fails to produce extracellular matrix and TGFβ and BMP signals essential for endothelial cell transformation and invasion of cardiac neural crest. We conclude that an autocrine regulatory loop, initiated by the reception of FGF signals by the mesoderm, regulates correct morphogenesis at the arterial pole of the heart. These findings provide new insight into how FGF signaling regulates context-dependent cellular responses during development.
An essential role for frizzled 5 in mammalian ocular development
Authors: Liu Chunqiao , Nathans Jeremy
Published: October 2008

Microphthalmia, coloboma and persistent fetal vasculature within the vitreous cavity are among the most common human congenital ocular anomalies,and each has been associated with a variety of genetic disorders. Here we show that, in the mouse, loss of frizzled 5 (Fz5) - a putative Wnt receptor expressed in the eye field, optic cup and retina - causes all of these defects with high penetrance. In the developing Fz5-/- eye, the sequence of defects, in order of appearance, is: increased cell death in the ventral retina, delayed and/or incomplete closure of the ventral fissure, an excess of mesenchymal cells in the vitreous cavity, an excess of retinal astrocyte precursors and mature astrocytes, and persistence of the hyaloid vasculature in association with a large number of pigment cells. Fz5-/- mice also exhibit a late-onset progressive retinal degeneration by ∼6 months of age, which might be related to the expression of Fz5 in Müller glia in the adult retina. These results demonstrate a central role for frizzled signaling in mammalian eye development and are likely to be relevant to the etiology of congenital human ocular anomalies.
Newborn Screening for Sickle Cell Disease Using Tandem Mass Spectrometry
Authors: Boemer François , Ketelslegers Olivier , Minon Jean-Marc , Bours Vincent , Schoos Roland
Published: October 2008

Background: Neonatal screening programs for sickle cell disease are now widespread in North American and European countries. Most programs apply isoelectric focusing or HPLC to detect hemoglobin variants. Because tandem mass spectrometry (MS/MS) is being used for screening of inherited metabolic disorders and allows protein identification, it was worth testing for hemoglobinopathy screening.

Methods: We minimized sample preparation and analysis times by avoiding prior purification, derivatization, or separation. We developed a tryptic digestion methodology to screen for the main clinically important variants (Hb S, Hb C, and Hb E) and β-thalassemia. To ensure proper discrimination between homozygote and heterozygote variants, we selected 4 transitions with good signal intensities for each specific peptide and calculated variant/Hb A ratios for each. Method validation included intra- and interseries variability, carryover, and limit of detection. We also performed a comparative study with isoelectric focusing results on 2082 specimens.

Results: Intraassay imprecision values (CVs) varied between 2.5% and 30.7%. Interassay CVs were between 6.3% and 23.6%. Carryover was <0.03%, and the limit of detection was fixed at 1% of Hb S. According to the MS/MS settings (detection of Hb S, Hb C, Hb E, and β-globin production defects), the comparative study did not yield any discrepant results between the 2 techniques.

Conclusions: MS/MS is a reliable method for hemoglobinopathy neonatal screening.
Seminal Oligouridinosis: Low Uridine Secretion as a Biomarker for Infertility in Spinal Neurotrauma
Authors: Maher Anthony D , Patki Prasad , Lindon John C , Want Elizabeth J , Holmes Elaine , Craggs Michael , Nicholson Jeremy K
Published: October 2008

Background: Compromised sexual health is a major rehabilitative barrier for men with lower–spinal cord injury (SCI). Although studies have revealed decreased sperm motility, the quantitative biochemical changes that underlie the infertility mechanism remain poorly understood.

Methods: We employed a nontargeted approach combining 800 MHz hydrogen nuclear magnetic resonance (1H NMR) spectroscopy and ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) with pattern recognition methods to analyze seminal fluid metabolite profiles in 10 men with and 8 without SCI above thoracic vertebra 10 (T10).

Results: The metabolic phenotype for SCI could be predicted from the 1H NMR data. The median concentration of uridine in fertile controls was 1.55 mmol/L (range 1.0–5.0 mmol/L), but was undetectable by both NMR and MS in all but 2 individuals from the SCI group, one who later fathered a child without assisted fertility techniques.

Conclusions: We hypothesize that uridine is likely to be an essential precursor to metabolites required for capacitation and is a potential marker for the prognosis of post-SCI functional fertility recovery. We derived the term “seminal oligouridinosis” to describe this newly identified condition.
Overexpression Screen in Drosophila Identifies Neuronal Roles of GSK-3β/shaggyas a Regulator of AP-1-Dependent Developmental Plasticity
Authors: Franciscovich A L , Mortimer A D Vrailas , Freeman A A , Gu J , Sanyal S
Published: October 2008

AP-1, an immediate-early transcription factor comprising heterodimers of the Fos and Jun proteins, has been shown in several animal models, including Drosophila, to control neuronal development and plasticity. In spite of this important role, very little is known about additional proteins that regulate, cooperate with, or are downstream targets of AP-1 in neurons. Here, we outline results from an overexpression/misexpression screen in Drosophila to identify potential regulators of AP-1 function at third instar larval neuromuscular junction (NMJ) synapses. First, we utilize >4000 enhancer and promoter (EP) and EPgy2 lines to screen a large subset of Drosophila genes for their ability to modify an AP-1-dependent eye-growth phenotype. Of 303 initially identified genes, we use a set of selection criteria to arrive at 25 prioritized genes from the resulting collection of putative interactors. Of these, perturbations in 13 genes result in synaptic phenotypes. Finally, we show that one candidate, the GSK-3β-kinase homolog, shaggy, negatively influences AP-1-dependent synaptic growth, by modulating the Jun-N-terminal kinase pathway, and also regulates presynaptic neurotransmitter release at the larval neuromuscular junction. Other candidates identified in this screen provide a useful starting point to investigate genes that interact with AP-1 in vivo to regulate neuronal development and plasticity.
Chromosome-Scale Genetic Mapping Using a Set of 16 Conditionally Stable Saccharomyces cerevisiae Chromosomes
Authors: Reid Robert J D , Sunjevaric Ivana , Voth Warren P , Ciccone Samantha , Du Wendy , Olsen Aileen E , Stillman David J , Rothstein Rodney
Published: October 2008

We have created a resource to rapidly map genetic traits to specific chromosomes in yeast. This mapping is done using a set of 16 yeast strains each containing a different chromosome with a conditionally functional centromere. Conditional centromere function is achieved by integration of a GAL1 promoter in cis to centromere sequences. We show that the 16 yeast chromosomes can be individually lost in diploid strains, which become hemizygous for the destabilized chromosome. Interestingly, most 2n − 1 strains endoduplicate and become 2n. We also demonstrate how chromosome loss in this set of strains can be used to map both recessive and dominant markers to specific chromosomes. In addition, we show that this method can be used to rapidly validate gene assignments from screens of strain libraries such as the yeast gene disruption collection.
Clonal Interference, Multiple Mutations and Adaptation in Large Asexual Populations
Authors: Fogle Craig A , Nagle James L , Desai Michael M
Published: October 2008

Two important problems affect the ability of asexual populations to accumulate beneficial mutations and hence to adapt. First, clonal interference causes some beneficial mutations to be outcompeted by more-fit mutations that occur in the same genetic background. Second, multiple mutations occur in some individuals, so even mutations of large effect can be outcompeted unless they occur in a good genetic background that contains other beneficial mutations. In this article, we use a Monte Carlo simulation to study how these two factors influence the adaptation of asexual populations. We find that the results depend qualitatively on the shape of the distribution of the fitness effects of possible beneficial mutations. When this distribution falls off slower than exponentially, clonal interference alone reasonably describes which mutations dominate the adaptation, although it gives a misleading picture of the evolutionary dynamics. When the distribution falls off faster than exponentially, an analysis based on multiple mutations is more appropriate. Using our simulations, we are able to explore the limits of validity of both of these approaches, and we explore the complex dynamics in the regimes where neither one is fully applicable.
Identification of Novel Regulators of atonal Expression in the Developing Drosophila Retina
Authors: Melicharek David , Shah Arpit , DiStefano Ginnene , Gangemi Andrew J , Orapallo Andrew , Vrailas-Mortimer Alysia D , Marenda Daniel R
Published: October 2008

Atonal is a Drosophila proneural protein required for the proper formation of the R8 photoreceptor cell, the founding photoreceptor cell in the developing retina. Proper expression and refinement of the Atonal protein is essential for the proper formation of the Drosophila adult eye. In vertebrates, expression of transcription factors orthologous to Drosophila Atonal (MATH5/Atoh7, XATH5, and ATH5) and their progressive restriction are also involved in specifying the retinal ganglion cell, the founding neural cell type in the mammalian retina. Thus, identifying factors that are involved in regulating the expression of Atonal during development are important to fully understand how retinal neurogenesis is accomplished. We have performed a chemical mutagenesis screen for autosomal dominant enhancers of a loss-of-function atonal eye phenotype. We report here the identification of five genes required for proper Atonal expression, three of which are novel regulators of Atonal expression in the Drosophila retina. We characterize the role of the daughterless, kismet, and roughened eye genes on atonal transcriptional regulation in the developing retina and show that each gene regulates atonal transcription differently within the context of retinal development. Our results provide additional insights into the regulation of Atonal expression in the developing Drosophila retina.
Selective Sweep at a Quantitative Trait Locus in the Presence of Background Genetic Variation
Authors: Chevin Luis-Miguel , Hospital Frédéric
Published: October 2008

We model selection at a locus affecting a quantitative trait (QTL) in the presence of genetic variance due to other loci. The dynamics at the QTL are related to the initial genotypic value and to the background genetic variance of the trait, assuming that background genetic values are normally distributed, under three different forms of selection on the trait. Approximate dynamics are derived under the assumption of small mutation effect. For similar strengths of selection on the trait (i.e, gradient of directional selection β) the way background variation affects the dynamics at the QTL critically depends on the shape of the fitness function. It generally causes the strength of selection on the QTL to decrease with time. The resulting neutral heterozygosity pattern resembles that of a selective sweep with a constant selection coefficient corresponding to the early conditions. The signature of selection may also be blurred by mutation and recombination in the later part of the sweep. We also study the race between the QTL and its genetic background toward a new optimum and find the conditions for a complete sweep. Overall, our results suggest that phenotypic traits exhibiting clear-cut molecular signatures of selection may represent a biased subset of all adaptive traits.
The Temporal Program of Chromosome Replication: Genomewide Replication inclb5ΔSaccharomyces cerevisiae
Authors: McCune Heather J , Danielson Laura S , Alvino Gina M , Collingwood David , Delrow Jeffrey J , Fangman Walton L , Brewer Bonita J , Raghuraman M K
Published: October 2008

Temporal regulation of origin activation is widely thought to explain the pattern of early- and late-replicating domains in the Saccharomyces cerevisiae genome. Recently, single-molecule analysis of replication suggested that stochastic processes acting on origins with different probabilities of activation could generate the observed kinetics of replication without requiring an underlying temporal order. To distinguish between these possibilities, we examined a clb5Δ strain, where origin firing is largely limited to the first half of S phase, to ask whether all origins nonspecifically show decreased firing (as expected for disordered firing) or if only some origins (“late” origins) are affected. Approximately half the origins in the mutant genome show delayed replication while the remainder replicate largely on time. The delayed regions can encompass hundreds of kilobases and generally correspond to regions that replicate late in wild-type cells. Kinetic analysis of replication in wild-type cells reveals broad windows of origin firing for both early and late origins. Our results are consistent with a temporal model in which origins can show some heterogeneity in both time and probability of origin firing, but clustering of temporally like origins nevertheless yields a genome that is organized into blocks showing different replication times.
Disruption of Rpn4-Induced Proteasome Expression in Saccharomyces cerevisiae Reduces Cell Viability Under Stressed Conditions
Authors: Wang Xiaogang , Xu Haiming , Ju Donghong , Xie Youming
Published: October 2008

The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the transcription activator Rpn4 upregulates the proteasome genes and is rapidly degraded by the assembled proteasome. Previous studies have shown that rpn4Δ cells are sensitive to a variety of stresses. However, the contribution of the loss of Rpn4-induced proteasome expression to the rpn4Δ phenotypes remains unclear because Rpn4 controls numerous genes other than the proteasome genes. Here we construct a yeast strain in which one of the essential proteasome genes, PRE1, is no longer induced by Rpn4. We show that the active proteasome level is lower in this strain than in the wild-type counterpart. Moreover, we demonstrate that loss of Rpn4-induced proteasome expression leads to cell-cycle delay in G2/M and sensitizes cells to various stresses. To our knowledge, this is the first report that explicitly reveals the physiological function of Rpn4-induced proteasome expression. This study also provides a tool for understanding the interactions between proteasome homeostasis and other cellular processes.
The EGL-4 PKG Acts With KIN-29 Salt-Inducible Kinase and Protein Kinase A to Regulate Chemoreceptor Gene Expression and Sensory Behaviors inCaenorhabditis elegans
Authors: van der Linden Alexander M , Wiener Scott , You Young-jai , Kim Kyuhyung , Avery Leon , Sengupta Piali
Published: October 2008

The regulation of chemoreceptor (CR) gene expression by environmental signals and internal cues may contribute to the modulation of multiple physiological processes and behavior in Caenorhabditis elegans. We previously showed that KIN-29, a homolog of salt-inducible kinase, acts in sensory neurons to regulate the expression of a subset of CR genes, as well as sensory behaviors. Here we show that the cGMP-dependent protein kinase EGL-4 acts partly in parallel with KIN-29 to regulate CR gene expression. Sensory inputs inhibit both EGL-4 and KIN-29 functions, and KIN-29 function is inhibited in turn by cAMP-dependent protein kinase (PKA) activation. EGL-4 and KIN-29 regulate CR gene expression by antagonizing the gene repression functions of the class II HDAC HDA-4 and the MEF-2 transcription factor, and KIN-29, EGL-4, and PKA target distinct residues in HDA-4 to regulate its function and subcellular localization. While KIN-29 acts primarily via MEF-2/HDA-4 to regulate additional sensory signal-regulated physiological processes and behaviors, EGL-4 acts via both MEF-2-dependent and -independent pathways. Our results suggest that integration of complex sensory inputs via multiple signaling pathways allows animals to precisely regulate sensory gene expression, thereby appropriately modulating physiology and behavior.
The Spindle Positioning Protein Kar9p Interacts With the Sumoylation Machinery in Saccharomyces cerevisiae
Authors: Meednu Nida , Hoops Harold , D'Silva Sonia , Pogorzala Leah , Wood Schuyler , Farkas David , Sorrentino Mark , Sia Elaine , Meluh Pam , Miller Rita K
Published: October 2008

Accurate positioning of the mitotic spindle is important for the genetic material to be distributed evenly in dividing cells, but little is known about the mechanisms that regulate this process. Here we report that two microtubule-associated proteins important for spindle positioning interact with several proteins in the sumoylation pathway. By two-hybrid analysis, Kar9p and Bim1p interact with the yeast SUMO Smt3p, the E2 enzyme Ubc9p, an E3 Nfi1p, as well as Wss1p, a weak suppressor of a temperature-sensitive smt3 allele. The physical interaction between Kar9p and Ubc9p was confirmed by in vitro binding assays. A single-amino-acid substitution in Kar9p, L304P disrupted its two-hybrid interaction with proteins in the sumoylation pathway, but retained its interactions with the spindle positioning proteins Bim1p, Stu2p, Bik1p, and Myo2p. The kar9-L304P mutant showed defects in positioning the mitotic spindle, with the spindle located more distally than normal. Whereas wild-type Kar9p-3GFP normally localizes to only the bud-directed spindle pole body (SPB), Kar9p-L304P-3GFP was mislocalized to both SPBs. Using a reconstitution assay, Kar9p was sumoylated in vitro. We propose a model in which sumoylation regulates spindle positioning by restricting Kar9p to one SPB. These findings raise the possibility that sumoylation could regulate other microtubule-dependent processes.