bluuebunny/crossref_metadata_2025 huggingface dataset:
Estimating uncertainty in observational studies of associations between continuous variables: example of methylmercury and neuropsychological testing in children
Authors: Goodman Michael , Barraj Leila M , Mink Pamela J , Britton Nicole L , Yager Janice W , Dana Flanders W , Kelsh Michael A
Published: September 2007

Background:

We suggest that the need to account for systematic error may explain the apparent lack of agreement among studies of maternal dietary methylmercury exposure and neuropsychological testing outcomes in children, a topic of ongoing debate.

Methods:

These sensitivity analyses address the possible role of systematic error on reported associations between low-level prenatal exposure to methylmercury and neuropsychological test results in two well known, but apparently conflicting cohort studies: the Faroe Islands Study (FIS) and the Seychelles Child Development Study (SCDS). We estimated the potential impact of confounding, selection bias, and information bias on reported results in these studies using the Boston Naming Test (BNT) score as the outcome variable.

Results:

Our findings indicate that, assuming various degrees of bias (in either direction) the corrected regression coefficients largely overlap. Thus, the reported effects in the two studies are not necessarily different from each other.

Conclusion:

Based on our sensitivity analysis results, it is not possible to draw definitive conclusions about the presence or absence of neurodevelopmental effects due to in utero methylmercury exposure at levels reported in the FIS and SCDS.
Recent changes in breast cancer incidence and risk factor prevalence in San Francisco Bay area and California women: 1988 to 2004
Authors: Keegan Theresa HM , Chang Ellen T , John Esther M , Horn-Ross Pamela L , Wrensch Margaret R , Glaser Sally L , Clarke Christina A
Published: September 2007

Introduction

Historically, the incidence rate of breast cancer among non-Hispanic white women living in the San Francisco Bay area (SFBA) of California has been among the highest in the world. Substantial declines in breast cancer incidence rates have been documented in the United States and elsewhere during recent years. In light of these reports, we examined recent changes in breast cancer incidence and risk factor prevalence among non-Hispanic white women in the SFBA and other regions of California.

Methods

Annual age-adjusted breast cancer incidence and mortality rates (1988 to 2004) were obtained from the California Cancer Registry and analyzed using Joinpoint regression. Population-based risk factor prevalences were calculated using two data sources: control subjects from four case-control studies (1989 to 1999) and the 2001 and 2003 California Health Interview Surveys.

Results

In the SFBA, incidence rates of invasive breast cancer increased 1.3% per year (95% confidence interval [CI], 0.7% to 2.0%) in 1988–1999 and decreased 3.6% per year (95% CI, 1.6% to 5.6%) in 1999–2004. In other regions of California, incidence rates of invasive breast cancer increased 0.8% per year (95% CI, 0.4% to 1.1%) in 1988–2001 and decreased 4.4% per year (95% CI, 1.4% to 7.3%) in 2001–2004. In both regions, recent (2000–2001 to 2003–2004) decreases in invasive breast cancer occurred only in women 40 years old or older and in women with all histologic subtypes and tumor sizes, hormone receptor-defined types, and all stages except distant disease. Mortality rates declined 2.2% per year (95% CI, 1.8% to 2.6%) from 1988 to 2004 in the SFBA and the rest of California. Use of estrogen-progestin hormone therapy decreased significantly from 2001 to 2003 in both regions. In 2003–2004, invasive breast cancer incidence remained higher (4.2%) in the SFBA than in the rest of California, consistent with the higher distributions of many established risk factors, including advanced education, nulliparity, late age at first birth, and alcohol consumption.

Conclusion

Ongoing surveillance of breast cancer occurrence patterns in this high-risk population informs breast cancer etiology through comparison of trends with lower-risk populations and by highlighting the importance of examining how broad migration patterns influence the geographic distribution of risk factors.
Autocrine WNT signaling contributes to breast cancer cell proliferation via the canonical WNT pathway and EGFR transactivation
Authors: Schlange Thomas , Matsuda Yutaka , Lienhard Susanne , Huber Alexandre , Hynes Nancy E
Published: September 2007

Background

De-regulation of the wingless and integration site growth factor (WNT) signaling pathway via mutations in APC and Axin, proteins that target β-catenin for destruction, have been linked to various types of human cancer. These genetic alterations rarely, if ever, are observed in breast tumors. However, various lines of evidence suggest that WNT signaling may also be de-regulated in breast cancer. Most breast tumors show hypermethylation of the promoter region of secreted Frizzled-related protein 1 (sFRP1), a negative WNT pathway regulator, leading to downregulation of its expression. As a consequence, WNT signaling is enhanced and may contribute to proliferation of human breast tumor cells. We previously demonstrated that, in addition to the canonical WNT/β-catenin pathway, WNT signaling activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mouse mammary epithelial cells via epidermal growth factor receptor (EGFR) transactivation.

Methods

Using the WNT modulator sFRP1 and short interfering RNA-mediated Dishevelled (DVL) knockdown, we interfered with autocrine WNT signaling at the ligand-receptor level. The impact on proliferation was measured by cell counting, YOPRO, and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay; β-catenin, EGFR, ERK1/2 activation, and PARP (poly [ADP-ribose]polymerase) cleavages were assessed by Western blotting after treatment of human breast cancer cell lines with conditioned media, purified proteins, small-molecule inhibitors, or blocking antibodies.

Results

Phospho-DVL and stabilized β-catenin are present in many breast tumor cell lines, indicating autocrine WNT signaling activity. Interfering with this loop decreases active β-catenin levels, lowers ERK1/2 activity, blocks proliferation, and induces apoptosis in MDA-MB-231, BT474, SkBr3, JIMT-1, and MCF-7 cells. The effects of WNT signaling are mediated partly by EGFR transactivation in human breast cancer cells in a metalloprotease- and Src-dependent manner. Furthermore, Wnt1 rescues estrogen receptor-positive (ER+) breast cancer cells from the anti-proliferative effects of 4-hydroxytamoxifen (4-HT) and this activity can be blocked by an EGFR tyrosine kinase inhibitor.

Conclusion

Our data show that interference with autocrine WNT signaling in human breast cancer reduces proliferation and survival of human breast cancer cells and rescues ER+ tumor cells from 4-HT by activation of the canonical WNT pathway and EGFR transactivation. These findings suggest that interference with WNT signaling at the ligand-receptor level in combination with other targeted therapies may improve the efficiency of breast cancer treatments.
Reversible and permanent effects of tobacco smoke exposure on airway epithelial gene expression
Authors: Beane Jennifer , Sebastiani Paola , Liu Gang , Brody Jerome S , Lenburg Marc E , Spira Avrum
Published: September 2007

Background

Tobacco use remains the leading preventable cause of death in the US. The risk of dying from smoking-related diseases remains elevated for former smokers years after quitting. The identification of irreversible effects of tobacco smoke on airway gene expression may provide insights into the causes of this elevated risk.

Results

Using oligonucleotide microarrays, we measured gene expression in large airway epithelial cells obtained via bronchoscopy from never, current, and former smokers (n = 104). Linear models identified 175 genes differentially expressed between current and never smokers, and classified these as irreversible (n = 28), slowly reversible (n = 6), or rapidly reversible (n = 139) based on their expression in former smokers. A greater percentage of irreversible and slowly reversible genes were down-regulated by smoking, suggesting possible mechanisms for persistent changes, such as allelic loss at 16q13. Similarities with airway epithelium gene expression changes caused by other environmental exposures suggest that common mechanisms are involved in the response to tobacco smoke. Finally, using irreversible genes, we built a biomarker of ever exposure to tobacco smoke capable of classifying an independent set of former and current smokers with 81% and 100% accuracy, respectively.

Conclusion

We have categorized smoking-related changes in airway gene expression by their degree of reversibility upon smoking cessation. Our findings provide insights into the mechanisms leading to reversible and persistent effects of tobacco smoke that may explain former smokers increased risk for developing tobacco-induced lung disease and provide novel targets for chemoprophylaxis. Airway gene expression may also serve as a sensitive biomarker to identify individuals with past exposure to tobacco smoke.
Aberrant dendritic cell differentiation initiated by theMll-Eenfusion gene does not require leukemic transformation
Authors: Sun Q , Kong C T , Huang F P , Chan L C
Published: September 2007

Dendritic cells (DCs), as specialized APCs, play a key role in the induction of anti-tumor immunity. They originate from bone marrow (BM) progenitors, which are frequently the targets of chromosomal translocations leading to development of leukemia. Aberrant DC differentiation and functions have been observed and are widely reported in patients with leukemia. It is not clear, however, whether such defects are a direct effect of a leukemic fusion gene or simply an outcome of the clinical disease. In this study, we demonstrate for the first time that knockin of the Mll-Een fusion gene can affect myeloid DC differentiation and functions directly, independent of the leukemic disease activities. We showed that the Mll-Een-expressing BM cells [enhanced green fluorescent protein+ (EGFP+)] from leukemic and nonleukemic mice had similarly impaired DC differentiation capacities with functional abnormalities. In contrast, BM cells without Mll-Een expression (EGFP−) showed normal DC differentiation and functions. A reduction in the frequency of CD11c+ DCs was also observed within the EGFP+ population in spleen and lymph nodes, and these cells were dysfunctional. Taken together, our findings suggest that the Mll-Een fusion gene can affect myeloid DC differentiation directly and functions in a cell-autonomous manner, where fully leukemic transformation of the hematopoietic progenitors is not required exclusively. Therefore, the study provides evidence for a direct causal relationship between leukemic gene fusion and abnormal DC differentiation, possibly contributing to the development of leukemia.
Adding Long‐acting β‐agonists to Inhaled Corticosteroids after Discharge from the Emergency Department for Acute Asthma: A Randomized Controlled Trial
Authors: Rowe Brian H. , Wong Eric , Blitz Sandra , Diner Barry , Mackey Duncan , Ross Scott , Senthilselvan Ambikaipakan
Published: September 2007

Background: Relapses of asthma following emergency department discharge can be reduced with oral and inhaled corticosteroids (ICSs), but the benefits of long‐acting β‐agonists (LABAs) are unclear.

Objectives: To determine whether the addition of a LABA reduces relapses in patients with acute asthma.

Methods: This was a randomized, controlled, double‐blind trial of 137 patients, aged 18–55 years, conducted in four Canadian EDs. Patients receiving high‐dose ICSs or oral corticosteroids, and those who were medically unstable, were excluded. Patients were randomized to either fluticasone 1,000 μg/day with salmeterol 100 μg/day or fluticasone 1,000 μg/day alone. All patients were discharged on seven days of oral prednisone. The main outcome measure was relapse at 21 days.

Results: Both groups had similar baseline characteristics. After 21 days, seven of 69 patients (10.1%) treated with fluticasone/salmeterol and ten of 68 patients (14.7%) treated with fluticasone experienced a relapse (p = 0.42). Prior intubation, female gender, and prior use of ICSs were associated with relapse. There were no clinically or statistically significant differences in overall quality of life and individual domain scores. Fluticasone/salmeterol improved quality of life (p < 0.05) and relapses (24% to 13%; p = 0.35) in patients receiving ICSs at the time of emergency admission.

Conclusions: Outpatient treatment with a short course of systemic corticosteroids combined with ICSs is adequate for most patients with asthma discharged from the emergency department; those already receiving ICS agents may benefit from ICS/LABA combination therapy to improve quality of life. Larger studies are needed to confirm the role of inhaled LABAs in acute asthma.
Training Retention of Level C Personal Protective Equipment Use by Emergency Medical Services Personnel
Authors: Northington William E. , Mahoney G. Michael , Hahn Michael E. , Suyama Joe , Hostler Dave
Published: September 2007

Objectives: To assess the six‐month training retention for out‐of‐hospital providers donning and doffing Level C personal protective equipment (PPE).

Methods: In this prospective observational study, 36 out‐of‐hospital providers enrolled in a paramedic program were trained in Level C (chemical‐resistant coverall, butyl gloves, and boots and an air‐purifying respirator) PPE use. A standardized training module and checklist of critical actions developed by a hazardous materials (hazmat) technician were used to evaluate donning and doffing. Students were trained until they were able to correctly don and doff the Level C PPE. An investigator used the checklist accompanying the training module to assess proficiency and remediate mistakes. Six months after initial training, the subjects were reassessed using the same investigator and checklist. Errors were designated as either critical (resulted in major self‐contamination of the airway, such as early removal of the respirator) or noncritical (potentially resulted in minor self‐contamination not involving the airway).

Results: Only five subjects (14.3%) were able to don and doff PPE without committing a critical error. The most common critical errors were premature removal of the respirator (65.7%; n= 23) and actions allowing the contaminated suit to touch the body (54.3%; n= 19). The most common noncritical error was possible self‐contamination due to the boots not being removed before exposing other body parts (37.1%; n= 13). Of the seven subjects (20%) with additional prior hazmat training, only two donned and doffed PPE without committing a critical error.

Conclusions: Retention of proper donning and doffing techniques in paramedic students is poor at six months after initial training. Even in subjects with previous hazmat, firefighter, and emergency medical services training, critical errors were common, suggesting that current training may be inadequate to prevent harmful exposures in emergency medical services personnel working at a hazmat or weapons of mass destruction incident.
National Study on the Quality of Emergency Department Care in the Treatment of Acute Myocardial Infarction and Pneumonia
Authors: Pham Julius Cuong , Kelen Gabor D. , Pronovost Peter J.
Published: September 2007

Objectives: To measure the quality of emergency department (ED) care for patients with acute myocardial infarction (AMI) and pneumonia (PNA) and to estimate the number of preventable deaths in these patients.

Methods: The authors performed a cross sectional study of ED visits with the diagnosis of AMI or PNA from 1998 to 2004. Data from the National Hospital Ambulatory Medical Care Survey were used. The study involved 544 EDs across the United States. The authors measured the proportion of patients receiving recommended therapies for AMI (aspirin and β‐blockers [BBs]) and PNA (appropriate antibiotics and pulse oximetry). The excess deaths associated with current care were estimated.

Results: Aspirin was administered to 40% and BBs to 17% of patients with AMI. Recommended antibiotics were administered to 69% and pulse oximetry was measured in 46% of patients with PNA. During the study period, the percentage receiving BBs and recommended antibiotics increased. There were more than 2.7 million opportunities to improve care and 22,000 excess deaths per year associated with current treatment of AMI and PNA.

Conclusions: Quality of care in the ED management of AMI and PNA is below national goals. This deficiency accounts for significant preventable deaths.
Communicating in the “Gray Zone”: Perceptions about Emergency Physician–hospitalist Handoffs and Patient Safety
Authors: Apker Julie , Mallak Larry A. , Gibson Scott C.
Published: September 2007

Objectives: To identify the perceptions of emergency physicians (EPs) and hospitalists regarding interservice handoff communication as patients are transferred from the emergency department to the inpatient setting.

Methods: Investigators conducted individual interviews with 12 physicians (six EPs and six hospitalists). Data evaluation consisted of using the steps of constant comparative, thematic analysis.

Results: Physicians perceived handoff communication as a gray zone characterized by ambiguity about patients' conditions and treatment. Two major themes emerged regarding the handoff gray zone. The first theme, poor communication practices and conflicting communication expectations, presented barriers that exacerbated physicians' information ambiguity. Specifically, handoffs consisting of insufficient information, incomplete data, omissions, and faulty information flow exacerbated gray zone problems and may negatively affect patient outcomes. EPs and hospitalists had different expectations about handoffs, and those expectations influenced their interactions in ways that may result in communication breakdowns. The second theme illustrated how poor handoff communication contributes to boarding‐related patient safety threats for boarders and emergency department patients alike. Those interviewed talked about the systemic failures that lead to patient boarding and how poor handoffs exacerbated system flaws.

Conclusions: Handoffs between EPs and hospitalists both reflect and contribute to the ambiguity inherent in emergency medicine. Poor handoffs, consisting of faulty communication behaviors and conflicting expectations for information, contribute to patient boarding conditions that can pose safety threats. Pragmatic conclusions are drawn regarding physician–physician communication in patient transfers, and recommendations are offered for medical education.
Comparison of Teen Driver Fatality Rates by Vehicle Type in the United States
Authors: Trowbridge Matthew J. , McKay Mary Pat , Maio Ronald F.
Published: September 2007

Objectives: To compare national fatality rates for teen drivers by vehicle type.

Methods: Fatality rates were calculated for 16‐ to 19‐year‐old drivers by vehicle type using data from the Fatal Analysis Reporting System (1999–2003) and estimates of miles driven from the National Household Transportation Survey (2001). Relative fatality risks for teen drivers of sports utility vehicles (SUVs) and pickups were calculated using passenger cars as a reference.

Results: Per vehicle mile driven, the fatality risk for both male and female teens driving SUVs was decreased relative to passenger car drivers (male teens: relative risk [RR], 0.33 [95% confidence interval [CI] = 0.29 to 0.37]; female teens: RR, 0.45 [95% CI = 0.34 to 0.59]). Fatality rates for male teens driving pickups were also lower per mile driven compared with male passenger car drivers (RR, 0.55 [95% CI = 0.51 to 0.60]). Fatality rates for female teens driving pickups and passenger cars were not statistically different but appear potentially higher for pickups (RR, 1.19 [95% CI = 0.98 to 1.44]). Both SUVs and pickups demonstrated significantly higher rates of fatal rollovers than passenger cars. Female adolescent drivers of SUVs and pickups were at particularly high risk for fatal rollovers per vehicle mile driven compared with passenger cars (SUV: RR, 1.88 [95% CI = 1.19 to 2.96]; pickup: RR, 3.42 [95% CI = 2.29 to 5.10]).

Conclusions: Fatality rates for teen drivers vary significantly by vehicle type. From 1999 to 2003 in the United States, fatal rollovers were significantly more likely per mile driven for teen drivers of both SUVs and pickups compared with passenger cars. However, overall fatality rates (i.e., all crash types) for teen drivers of SUVs and male drivers of pickups were lower per mile driven than for teen drivers of passenger cars. The results of this ecological analysis cannot predict the individual‐level fatality risk for teens driving different vehicle types. However, the significant variability in fatality rates among SUVs, pickups, and passenger cars seen at a population level suggests that vehicle choice should be further explored as a potentially modifiable risk factor in interventions to address teen driver safety.
Use of Ultrasound Measurement of the Inferior Vena Cava Diameter as an Objective Tool in the Assessment of Children with Clinical Dehydration
Authors: Chen Lei , Kim Yunie , Santucci Karen A.
Published: September 2007

Objectives: Bedside ultrasonography (US) measurement of the inferior vena cava (IVC) and aorta (Ao) may be useful in objectively assessing children with dehydration. The objectives of this study were 1) to compare the IVC and Ao diameters (IVC/Ao) ratio of dehydrated children with controls and 2) to compare the IVC/Ao ratio before and after intravenous (IV) rehydration in children with dehydration.

Methods: This prospective observational study was performed in an urban pediatric emergency department. Children between 6 months and 16 years of age with clinical evidence of dehydration were enrolled. Bedside US measurements of the IVC and Ao were taken before and immediately after IV fluids were administered. An age‐, gender‐, and weight‐matched control without dehydration was enrolled for each subject. The IVC/Ao ratios of subjects and controls were compared using Wilcoxon signed rank test, as were the ratios before and after IV hydration for each subject.

Results: Thirty‐six pairs of subjects and matched controls were enrolled. The IVC/Ao ratios in the subjects were lower as compared with controls (mean of 0.75 vs. 1.01), with a mean difference of 0.26 (95% confidence interval = 0.18 to 0.35). In subjects, the IVC/Ao ratios were significantly lower before IV hydration (mean of 0.75 vs. 1.09), with a mean difference of 0.34 (95% confidence interval = 0.29 to 0.39).

Conclusions: As measured by bedside US measurement, the IVC/Ao ratio is lower in children clinically assessed to be dehydrated. Furthermore, it increases with administration of IV fluid boluses.
Promotion of lens epithelial-fiber differentiation by the C-terminus of connexin 45.6 – a role independent of gap junction communication
Authors: Banks Eric A. , Yu X. Sean , Shi Qian , Jiang Jean X.
Published: September 2007

We previously reported that, among the three connexins expressed in chick lens, overexpression of connexin (Cx) 45.6, not Cx43 or Cx56, stimulates lens cell differentiation; however, the underlying mechanism responsible for this effect is unclear. Here, we took advantage of naturally occurring loss-of-gap-junction function mutations of Cx50 (ortholog of chick Cx45.6) and generated the corresponding site mutants in Cx45.6: Cx45.6(D47A) and Cx45.6(P88S). In contrast to wild-type Cx45.6, the mutants failed to form functional gap junctions, and Cx45.6(P88S) and, to a lesser degree, Cx45.6(D47A) functioned in a dominant-negative manner. Interestingly, overexpression of both mutants incapable of forming gap junctions significantly increased epithelial-fiber differentiation to a level comparable to that of wild-type Cx45.6. To map the functional domain of Cx45.6, we generated a C-terminus chimera as well as deletion mutants. Overexpression of Cx56*45.6C, the mutant in which the C-terminus of Cx56 was replaced with that of Cx45.6, had a stimulatory effect on lens cell differentiation similar to that of Cx45.6. However, cells overexpressing Cx45.6*56C, the mutant in which C-terminus of Cx45.6 was replaced with that of Cx56, and Cx45.6(–C), in which the C-terminus was deleted, failed to promote differentiation. Taken together, we conclude that the expression of Cx45.6, but not Cx45.6-dependent gap junction channels, is involved in lens epithelial-fiber cell differentiation, and the C-terminal domain of Cx45.6 plays a predominant role in mediating this process.
DAP kinase mediates the phosphorylation of tropomyosin-1 downstream of the ERK pathway, which regulates the formation of stress fibers in response to oxidative stress
Authors: Houle François , Poirier Andrée , Dumaresq Jeannot , Huot Jacques
Published: September 2007

Endothelial cells are actively involved in regulating the exchanges between blood and tissues. This function is tightly dependent on actin cytoskeleton dynamics and is challenged by a wide variety of stimuli, including oxidative stress. In endothelial cells, oxidative stress quickly activates the extracellular-signal-regulated kinase (ERK) MAP kinase, which results in the phosphorylation of tropomyosin. Here, we investigated further the mechanisms of tropomyosin phosphorylation and its function in actin remodeling. We identified, for the first time, death-associated protein kinase 1 (DAP kinase 1) as the kinase that phosphorylates tropomyosin-1 in response to ERK activation by hydrogen peroxide (H2O2). We also report that the phosphorylation of tropomyosin-1 mediated by DAP kinase occurs on Ser283. Moreover, the expression of the pseudophosphorylated tropomyosin mutant Ser283Glu triggers by itself the formation of stress fibers in untreated cells, and the effect is maintained in H2O2-treated cells in which DAP kinase expression is knocked-down by siRNA. By contrast, the expression of the nonphosphorylatable tropomyosin mutant Ser283Ala is not associated with stress fibers and leads to membrane blebbing in response to H2O2. Our finding that tropomyosin-1 is phosphorylated downstream of ERK and DAP kinase and that it helps regulate the formation of stress fibers will aid understanding the role of this protein in regulating the endothelial functions associated with cytoskeletal remodeling.
The ADAMTS12 metalloproteinase exhibits anti-tumorigenic properties through modulation of the Ras-dependent ERK signalling pathway
Authors: Llamazares María , Obaya Alvaro J. , Moncada-Pazos Angela , Heljasvaara Ritva , Espada Jesús , López-Otín Carlos , Cal Santiago
Published: September 2007

Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteolytic enzymes are implicated in a variety of physiological processes, such as collagen maturation, organogenesis, angiogenesis, reproduction and inflammation. Moreover, deficiency or overexpression of certain ADAMTS proteins is directly involved in serious human diseases, including cancer. However, the functional roles of other family members, such as ADAMTS12, remain unknown. Here, by using different in vitro and in vivo approaches, we have evaluated the possible role of ADAMTS12 in the development and progression of cancer. First, we show that expression of ADAMTS12 in Madin-Darby canine kidney (MDCK) cells prevents the tumorigenic effects of hepatocyte growth factor (HGF) by blocking the activation of the Ras-MAPK signalling pathway and that this regulation involves the thrombospondin domains of the metalloproteinase. We also show that addition of recombinant human ADAMTS12 to bovine aortic endothelial cells (BAE-1 cells) abolishes their ability to form tubules upon stimulation with vascular endothelial growth factor (VEGF). Additionally, tumours induced in immunodeficient SCID mice injected with A549 cells overexpressing ADAMTS12 show a remarkable growth deficiency in comparison with tumours formed in animals injected with parental A549 cells. Overall, our data suggest that ADAMTS12 confers tumour-protective functions upon cells that produce this proteolytic enzyme.
Schizosaccharomyces pombeprotein phosphatase 1 in mitosis, endocytosis and a partnership with Wsh3/Tea4 to control polarised growth
Authors: Alvarez-Tabarés Isabel , Grallert Agnes , Ortiz Jose-Miguel , Hagan Iain M.
Published: September 2007

PP1 holoenzymes are composed of a small number of catalytic subunits and an array of regulatory, targeting, subunits. The Schizosaccharomyces pombe genome encodes two highly related catalytic subunits, Dis2 and Sds21. The gene for either protein can be individually deleted, however, simultaneous deletion of both is lethal. We fused enhanced green fluorescent protein (EGFP) coding sequences to the 5′ end of the endogenous sds21+ and dis2+ genes. Dis2.NEGFP accumulated in nuclei, associated with centromeres, foci at cell tips and endocytic vesicles. This actin-dependent endocytosis occurred between nuclei and growing tips and was polarised towards growing tips. When dis2+ was present, Sds21.NEGFP was predominantly a nuclear protein, greatly enriched in the nucleolus. When dis2+ was deleted, Sds21.NEGFP levels increased and Sds21.NEGFP was then clearly detected at centromeres, endocytic vesicles and cell tips. Dis2.NEGFP was recruited to cell tips by the formin binding, stress pathway scaffold Wsh3 (also known as Tea4). Wsh3/Tea4 modulates polarised tip growth in unperturbed cell cycles and governs polarised growth following osmotic stress. Mutating the PP1 recruiting RVXF motif in Wsh3/Tea4 blocked PP1 binding, altered cell cycle regulated growth to induce branching, induced branching from existing tips in response to stress, and blocked the induction of actin filaments that would otherwise arise from Wsh3/Tea4 overproduction.
EGF-receptor-mediated mammary epithelial cell migration is driven by sustained ERK signaling from autocrine stimulation
Authors: Joslin Elizabeth J. , Opresko Lee K. , Wells Alan , Wiley H. Steven , Lauffenburger Douglas A.
Published: September 2007

EGF family ligands are synthesized as membrane-anchored precursors whose proteolytic release yields mature diffusible factors that can activate cell surface receptors in autocrine or paracrine mode. Expression of these ligands is altered in pathological states and in physiological processes, such as development and tissue regeneration. Despite the widely documented biological importance of autocrine EGF signaling, quantitative relationships between protease-mediated ligand release and consequent cell behavior have not been rigorously investigated. We thus explored the relationship between autocrine EGF release rates and cell behavioral responses along with activation of ERK, a key downstream signal, by expressing chimeric ligand precursors and modulating their proteolytic shedding using a metalloprotease inhibitor in human mammary epithelial cells. We found that ERK activation increased monotonically with increasing ligand release rate despite concomitant downregulation of EGF receptor levels. Cell migration speed was directly related to ligand release rate and proportional to steady-state phospho-ERK levels. Moreover, migration speed was significantly greater for autocrine stimulation compared with exogenous stimulation, even at comparable phospho-ERK levels. By contrast, cell proliferation rates were approximately equivalent at all ligand release rates and were similar regardless of whether the ligand was presented endogenously or exogenously. Thus, in our mammary epithelial cell system, migration and proliferation are differentially sensitive to the mode of EGF ligand presentation.
Mitch – a rapidly evolving component of the Ndc80 kinetochore complex required for correct chromosome segregation inDrosophila
Authors: Williams Byron , Leung Garmay , Maiato Helder , Wong Alex , Li ZeXiao , Williams Erika V. , Kirkpatrick Catherine , Aquadro Charles F. , Rieder Conly L. , Goldberg Michael L.
Published: September 2007

We identified an essential kinetochore protein, Mitch, from a genetic screen in D. melanogaster. Mitch localizes to the kinetochore, and its targeting is independent of microtubules (MTs) and several other known kinetochore components. Animals carrying mutations in mitch die as late third-instar larvae; mitotic neuroblasts in larval brains exhibit high levels of aneuploidy. Analysis of fixed D. melanogaster brains and mitch RNAi in cultured cells, as well as video recordings of cultured mitch mutant neuroblasts, reveal that chromosome alignment in mitch mutants is compromised during spindle formation, with many chromosomes displaying persistent mono-orientation. These misalignments lead to aneuploidy during anaphase. Mutations in mitch also disrupt chromosome behavior during both meiotic divisions in spermatocytes: the entire chromosome complement often moves to only one spindle pole. Mutant mitotic cells exhibit contradictory behavior with respect to the spindle assembly checkpoint (SAC). Anaphase onset is delayed in untreated cells, probably because incorrect kinetochore attachment maintains the SAC. However, mutant brain cells and mitch RNAi cells treated with MT poisons prematurely disjoin their chromatids, and exit mitosis. These data suggest that Mitch participates in SAC signaling that responds specifically to disruptions in spindle microtubule dynamics. The mitch gene corresponds to the transcriptional unit CG7242, and encodes a protein that is a possible ortholog of the Spc24 or Spc25 subunit of the Ndc80 kinetochore complex. Despite the crucial role of Mitch in cell division, the mitch gene has evolved very rapidly among species in the genus Drosophila.
Chlamydomonas FAP133 is a dynein intermediate chain associated with the retrograde intraflagellar transport motor
Authors: Rompolas Panteleimon , Pedersen Lotte B. , Patel-King Ramila S. , King Stephen M.
Published: September 2007

Intraflagellar transport (IFT) is the bi-directional movement of particles along the length of axonemal outer doublet microtubules and is needed for the assembly and maintenance of eukaryotic cilia and flagella. Retrograde IFT requires cytoplasmic dynein 1b, a motor complex whose organization, structural composition and regulation is poorly understood. We have characterized the product of the Chlamydomonas FAP133 gene that encodes a new WD-repeat protein similar to dynein intermediate chains and homologous to the uncharacterized vertebrate protein WD34. FAP133 is located at the peri-basal body region as well as in punctate structures along the flagella. This protein is associated with the IFT machinery because it is specifically depleted from the flagella of cells with defects in anterograde IFT. Fractionation of flagellar matrix proteins indicates that FAP133 associates with both the LC8 dynein light chain and the IFT dynein heavy chain and light intermediate chain (DHC1b-D1bLIC) motor complex. In the absence of DHC1b or D1bLIC, FAP133 fails to localize at the peri-basal body region but, rather, is concentrated in a region of the cytoplasm near the cell center. Furthermore, we found that FAP133, LC8, DHC1b, D1bLIC, the FLA10 kinesin-2 necessary for anterograde IFT and other IFT scaffold components associate to form a large macromolecular assembly.
Microcephalin coordinates mitosis in the syncytialDrosophilaembryo
Authors: Brunk Kathrin , Vernay Bertrand , Griffith Elen , Reynolds Natalie L. , Strutt David , Ingham Philip W. , Jackson Andrew P.
Published: September 2007

Microcephalin (MCPH1) is mutated in primary microcephaly, an autosomal recessive human disorder of reduced brain size. It encodes a protein with three BRCT domains that has established roles in DNA damage signalling and the cell cycle, regulating chromosome condensation. Significant adaptive evolutionary changes in primate MCPH1 sequence suggest that changes in this gene could have contributed to the evolution of the human brain. To understand the developmental role of microcephalin we have studied its function in Drosophila. We report here that Drosophila MCPH1 is cyclically localised during the cell cycle, co-localising with DNA during interphase, but not with mitotic chromosomes. mcph1 mutant flies have a maternal effect lethal phenotype, due to mitotic arrest occurring in early syncytial cell cycles. Mitotic entry is slowed from the very first mitosis in such embryos, with prolonged prophase and metaphase stages; and frequent premature separation as well as detachment of centrosomes. As a consequence, centrosome and nuclear cycles become uncoordinated, resulting in arrested embryonic development. Phenotypic similarities with abnormal spindle (asp) and centrosomin (cnn) mutants (whose human orthologues are also mutated in primary microcephaly), suggest that further studies in the Drosophila embryo may establish a common developmental and cellular pathway underlying the human primary microcephaly phenotype.
TheDrosophilahomolog ofMCPH1,a human microcephaly gene, is required for genomic stability in the early embryo
Authors: Rickmyre Jamie L. , DasGupta Shamik , Ooi Danny Liang-Yee , Keel Jessica , Lee Ethan , Kirschner Marc W. , Waddell Scott , Lee Laura A.
Published: September 2007

Mutation of human microcephalin (MCPH1) causes autosomal recessive primary microcephaly, a developmental disorder characterized by reduced brain size. We identified mcph1, the Drosophila homolog of MCPH1, in a genetic screen for regulators of S-M cycles in the early embryo. Embryos of null mcph1 female flies undergo mitotic arrest with barrel-shaped spindles lacking centrosomes. Mutation of Chk2 suppresses these defects, indicating that they occur secondary to a previously described Chk2-mediated response to mitotic entry with unreplicated or damaged DNA. mcph1 embryos exhibit genomic instability as evidenced by frequent chromatin bridging in anaphase. In contrast to studies of human MCPH1, the ATR/Chk1-mediated DNA checkpoint is intact in Drosophila mcph1 mutants. Components of this checkpoint, however, appear to cooperate with MCPH1 to regulate embryonic cell cycles in a manner independent of Cdk1 phosphorylation. We propose a model in which MCPH1 coordinates the S-M transition in fly embryos: in the absence of mcph1, premature chromosome condensation results in mitotic entry with unreplicated DNA, genomic instability, and Chk2-mediated mitotic arrest. Finally, brains of mcph1 adult male flies have defects in mushroom body structure, suggesting an evolutionarily conserved role for MCPH1 in brain development.
Reversible interactions between smooth domains of the endoplasmic reticulum and mitochondria are regulated by physiological cytosolic Ca2+ levels
Authors: Goetz Jacky G. , Genty Hélène , St-Pierre Pascal , Dang Thao , Joshi Bharat , Sauvé Rémy , Vogl Wayne , Nabi Ivan R.
Published: September 2007

The 3F3A monoclonal antibody to autocrine motility factor receptor (AMFR) labels mitochondria-associated smooth endoplasmic reticulum (ER) tubules. siRNA down-regulation of AMFR expression reduces mitochondria-associated 3F3A labelling. The 3F3A-labelled ER domain does not overlap with reticulon-labelled ER tubules, the nuclear membrane or perinuclear ER markers and only partially overlaps with the translocon component Sec61α. Upon overexpression of FLAG-tagged AMFR, 3F3A labelling is mitochondria associated, excluded from the perinuclear ER and co-distributes with reticulon. 3F3A labelling therefore defines a distinct mitochondria-associated ER domain. Elevation of free cytosolic Ca2+ levels with ionomycin promotes dissociation of 3F3A-labelled tubules from mitochondria and, judged by electron microscopy, disrupts close contacts (<50 nm) between smooth ER tubules and mitochondria. The ER tubule-mitochondria association is similarly disrupted upon thapsigargin-induced release of ER Ca2+ stores or purinergic receptor stimulation by ATP. The inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] receptor (IP3R) colocalises to 3F3A-labelled mitochondria-associated ER tubules, and conditions that induce ER tubule-mitochondria dissociation disrupt continuity between 3F3A- and IP3R-labelled ER domains. RAS-transformed NIH-3T3 cells have increased basal cytosolic Ca2+ levels and show dissociation of the 3F3A-labelled, but not IP3R-labelled, ER from mitochondria. Our data indicate that regulation of the ER-mitochondria association by free cytosolic Ca2+ is a characteristic of smooth ER domains and that multiple mechanisms regulate the interaction between these organelles.
The subcellular localization of the Niemann-Pick Type C proteins depends on the adaptor complex AP-3
Authors: Berger Adam C. , Salazar Gloria , Styers Melanie L. , Newell-Litwa Karen A. , Werner Erica , Maue Robert A. , Corbett Anita H. , Faundez Victor
Published: September 2007

Niemann-Pick Type C (NP-C) disease, caused by mutations in either human NPC1 (hNPC1) or human NPC2 (hNPC2), is characterized by the accumulation of unesterified cholesterol in late endosomes. Although it is known that the NP-C proteins are targeted to late endosomal/lysosomal compartments, their delivery mechanisms have not been fully elucidated. To identify mechanisms regulating NP-C protein localization, we used Saccharomyces cerevisiae, which expresses functional homologs of both NP-C proteins – scNcr1p and scNpc2p. Targeting of scNcr1p to the vacuole was perturbed in AP-3-deficient yeast cells, whereas the delivery of scNpc2p was affected by deficiencies in either AP-3 or GGA. We focused on the role of the AP-3 pathway in the targeting of the mammalian NP-C proteins. We found that, although mouse NPC1 (mNPC1) and hNPC2 co-localize with AP-3 to a similar extent in fibroblasts, hNPC2 preferentially co-localizes with AP-1. Importantly, the targeting of both mammalian NPC1 and NPC2 is dependent on AP-3. Moreover, and consistent with the NP-C proteins playing a role in cholesterol metabolism, AP-3-deficient cells have reduced levels of cholesterol. These results provide information about how the NP-C proteins are targeted to their sites of action and illustrate the possibility that defective sorting of the NP-C proteins along the endocytic route can alter cellular cholesterol.
Selective quenching of fluorescence from unbound oligonucleotides by gold nanoparticles as a probe of RNA structure
Authors: Li Huixiang , Liang Ruiting , Turner Douglas H. , Rothberg Lewis J. , Duan Shenghua
Published: September 2007

Binding of small oligonucleotides to the periphery of folded RNA can provide insight into the secondary structure of complex RNA in solution. To discriminate between bound and unbound fluorescein-labeled 2′-O-methyl RNA probes, we use ionically coated gold nanoparticles to selectively adsorb unbound probes and quench their fluorescence. The target is the 3′ untranslated region of Bombyx mori R2 RNA. Fluorescence indicates that R2 sequences complementary to some of the probes are accessible for binding in the three-dimensional structure. Hybridization occurs under homogeneous conditions in the absence of the gold nanoparticles so that steric issues associated with chip-based assays are avoided. The assay is compatible with well plate formats, takes less than 5 min, and requires only 2 pmol or less of unlabeled target RNA per probe sequence tested.
Repetition of Deliberate Self‐Harm: A Study of the Characteristics and Subsequent Deaths in Patients Presenting to a General Hospital According to Extent of Repetition
Authors: Haw Camilla , Bergen Helen , Casey Deborah , Hawton Keith
Published: September 2007

Repetition of deliberate self‐harm (DSH) is common. Some patients repeat multiple times. We have investigated the characteristics of repeaters, and mortality in three groups of DSH patients by repetition status. Data collected by the Oxford Monitoring System for Attempted Suicide were used to examine the pattern of repetition of DSH patients presenting to a general hospital between 1990 and 1997. Each patient was tracked through the monitoring system with regard to repetition. Patients traceable through National Death Registers were followed up until 2000 with respect to mortality. A total of 4,167 patients were studied of which 1,022 (24.5%) repeated at least once during follow‐up. Using multinomial regression, past history of DSH was the variable most strongly associated with frequent (4+) and less frequent (1–3) repetitions. Risk of suicide was significantly increased in females with frequent repetition (7.7% dying by suicide), compared with both those with 1–3 repetitions (2.3%) and those not repeating (1.0%). The analyses were repeated for the 2,167 patients with no past history of DSH at their first presentation. Using multinomial regression, personality disorder was the only variable that was associated with 4+ repetitions compared with no repetitions, although a number of variables distinguished between patients with 1–3 repetitions and no repeat episodes. For clinicians assessing DSH patients, past history of DSH is the best predictor of infrequent and frequent repetition. In patients with no past history of DSH the presence of personality disorder increases the risk of frequent repetition of DSH.
I Hear, I Listen and I Care: A Qualitative Investigation into the Function of a Self‐Harm Message Board
Authors: Rodham Karen , Gavin Jeff , Miles Meriel
Published: September 2007

This paper reports on the findings of a qualitative inquiry into the interactions on a nonprofessional self‐harm message board. Individuals using the message board were very positive about the message board and appeared to feel that their needs for support, venting, and validation were being met. However, we found that negative harmful behaviors mentioned in messages posted on the board were either not acknowledged, or were legitimized and normalized by those who responded to the messages. We suggest that this process of minimizing the seriousness and/or normalizing the behaviors may actually be encouraging the maintenance of such behaviors and therefore conclude that caution should be exercised when using nonprofessional self‐harm message boards.
Coping Mechanisms, Stressful Events and Suicidal Behavior Among Youth Admitted to Juvenile Justice and Child Welfare Services
Authors: Chagnon François
Published: September 2007

This study examines the relationship between coping mechanisms and suicide attempts among 84 adolescents in the Quebec youth residential services. A new measure based on in‐depth interviews is used to identify coping mechanisms. The suicidal youths used fewer adequate coping mechanisms than nonsuicidal youths when faced with comparable stressful events. Negative cognitive reframing, anger, and blaming others were more frequently reported by the suicidal youths. Contrary to certain theoretical conceptions, coping mechanisms were not stable and varied according to the context of the suicidal crisis. These findings indicate the usefulness of this approach to measure coping mechanisms and support the importance of programs aimed at developing coping skills for suicide prevention.
Heroin Addicts Reporting Previous Heroin Overdoses Also Report Suicide Attempts
Authors: Brådvik Louise , Frank Arne , Hulenvik Per , Medvedeo Alvaro , Berglund Mats
Published: September 2007

Nonfatal heroin overdoses and suicide attempts are both common among heroin addicts, but there is limited knowledge about the association between them. The sample in the present study consisted of 149 regular heroin users in Malmö, Sweden. Out of these 98 had taken an unintentional heroin overdose at some time and 51 had made at least one attempt to commit suicide (but not using heroin). Suicide attempts were significantly more common among those who had taken unintentional overdoses as compared with those who had never taken any overdose (p < 0.01). The more overdoses, the greater the risk of suicide attempt.