Third Annual Report: 2006 Pre-Construction Eelgrass Monitoring and Propagation for King County Outfall Mitigation

King County proposes to build a new sewer outfall discharging to Puget Sound near Point Wells, Washington. Construction is scheduled for 2008. The Point Wells site was selected to minimize effects on the nearshore marine environment, but unavoidable impacts to eelgrass (Zostera marina) beds are anticipated during construction. To mitigate for these impacts and prepare for post-construction restoration, King County began implementation of a multi-year eelgrass monitoring and restoration program in 2004, with the primary goal of returning intertidal and shallow subtidal habitat and eelgrass to pre-construction conditions. Major program elements are a) pre-construction monitoring, i.e., documenting initial eelgrass conditions and degree of fluctuation over 5 years prior to construction, b) eelgrass transplanting, including harvesting, offsite propagating and stockpiling of local plantstock, and post-construction planting, and c) post-construction monitoring. The program is detailed in the Eelgrass Restoration and Biological Resources Implementation Workplan (King County 2006). This report describes calendar year 2006 pre-construction activities conducted by Pacific Northwest National Laboratory (PNNL) in support of King County. Activities included continued propagation of eelgrass shoots and monitoring of the experimental harvest plots in the marine outfall corridor area to evaluate recovery rates relative to harvest rates. Approximately 1500 additional shoots were harvested from the marine outfall corridor in August 2006 to supplement the plants in the propagation tank at the PNNL Marine Sciences Laboratory in Sequim, Washington, bringing the total number of shoots to 4732. Eelgrass densities were monitored in the five experimental harvest plots established in the marine outfall corridor. Changes in eelgrass density were evaluated in year-to-year comparisons with initial harvest rates. Net eelgrass density decreased from 2004 post-harvest to 2006 in all plots, despite density increases observed in 2005 in some plots and at some harvest rates. Eelgrass densities within individual subplots were highly variable from year to year, and the change in density in any interannual period did not correlate to the initial 2004 harvest rate. Continued monitoring should help project managers determine an optimum harvest rate that supports rapid recovery of donor eelgrass beds

Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival

Recent evidence suggests CML stem cells are insensitive to kinase inhibitors and responsible for minimal residual disease in treated patients. We investigated whether CML stem cells, in a transgenic mouse model of CML-like disease or derived from patients, are dependent on Bcr-Abl. In the transgenic model, following re-transplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off, were able to persist in vivo and re-initiate leukemia in secondary recipients upon Bcr-Abl re-expression. Bcr-Abl knockdown in human CD34+ CML cells cultured for 12 days in physiological growth factors achieved partial inhibition of Bcr-Abl and downstream targets p-CrkL and p-STAT5, inhibition of proliferation and colony forming cells, but no reduction of input cells. The addition of dasatinib further inhibited p-CrkL and p-STAT5, yet only reduced input cells by 50%. Complete growth factor withdrawal plus dasatinib further reduced input cells to 10%, however the surviving fraction was enriched for primitive leukemic cells capable of growth in long-term culture initiating cell assay and expansion upon removal of dasatinib and addition of growth factors. Together these data suggest that CML stem cell survival is Bcr-Abl kinase independent and suggest curative approaches in CML must focus on kinase-independent mechanisms of resistance

Transmission electron microscopy characterization of fluorescently labelled amyloid β 1-40 and α-synuclein aggregates

<p>Abstract</p> <p>Background</p> <p>Fluorescent tags, including small organic molecules and fluorescent proteins, enable the localization of protein molecules in biomedical research experiments. However, the use of these labels may interfere with the formation of larger-scale protein structures such as amyloid aggregates. Therefore, we investigate the effects of some commonly used fluorescent tags on the morphologies of fibrils grown from the Alzheimer's disease-associated peptide Amyloid β 1-40 (Aβ40) and the Parkinson's disease-associated protein α-synuclein (αS).</p> <p>Results</p> <p>Using transmission electron microscopy (TEM), we verify that N-terminal labeling of Aβ40 with AMCA, TAMRA, and Hilyte-Fluor 488 tags does not prevent the formation of protofibrils and amyloid fibrils of various widths. We also measure the two-photon action cross-section of Aβ40 labelled with Hilyte Fluor 488 and demonstrate that this tag is suitable for use with two-photon fluorescence techniques. Similarly, we find that Alexa Fluor 488 labelling of αS variant proteins near either the N or C terminus (position 9 or 130) does not interfere with the formation of amyloid and other types of αS fibrils. We also present TEM images of fibrils grown from αS C-terminally labelled with enhanced green fluorescent protein (EGFP). Near neutral pH, two types of αS-EGFP fibrils are observed via TEM, while denaturation of the EGFP tag leads to the formation of additional species.</p> <p>Conclusions</p> <p>We demonstrate that several small extrinsic fluorescent tags are compatible with studies of amyloid protein aggregation. However, although fibrils can be grown from αS labelled with EGFP, the conformation of the fluorescent protein tag affects the observed aggregate morphologies. Thus, our results should assist researchers with label selection and optimization of solution conditions for aggregation studies involving fluorescence techniques.</p

Targeting Human Central Nervous System Protein Kinases: An Isoform Selective p38αMAPK Inhibitor that Attenuates Disease Progression in Alzheimer\u27s Disease Mouse Models

The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150\u27s exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior

Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction

Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction

College student sleep quality and mental and physical health are associated with food insecurity in a multi-campus study

Objective: To assess the relationship between food insecurity, sleep quality, and days with mental and physical health issues among college students. Design: An online survey was administered. Food insecurity was assessed using the ten-item Adult Food Security Survey Module. Sleep was measured using the nineteen-item Pittsburgh Sleep Quality Index (PSQI). Mental health and physical health were measured using three items from the Healthy Days Core Module. Multivariate logistic regression was conducted to assess the relationship between food insecurity, sleep quality, and days with poor mental and physical health. Setting: Twenty-two higher education institutions. Participants: College students (n 17 686) enrolled at one of twenty-two participating universities. Results: Compared with food-secure students, those classified as food insecure (43·4 %) had higher PSQI scores indicating poorer sleep quality (P \u3c 0·0001) and reported more days with poor mental (P \u3c 0·0001) and physical (P \u3c 0·0001) health as well as days when mental and physical health prevented them from completing daily activities (P \u3c 0·0001). Food-insecure students had higher adjusted odds of having poor sleep quality (adjusted OR (AOR): 1·13; 95 % CI 1·12, 1·14), days with poor physical health (AOR: 1·01; 95 % CI 1·01, 1·02), days with poor mental health (AOR: 1·03; 95 % CI 1·02, 1·03) and days when poor mental or physical health prevented them from completing daily activities (AOR: 1·03; 95 % CI 1·02, 1·04). Conclusions: College students report high food insecurity which is associated with poor mental and physical health, and sleep quality. Multi-level policy changes and campus wellness programmes are needed to prevent food insecurity and improve student health-related outcomes

Development of novel methods for non-canonical myeloma protein analysis with an innovative adaptation of immunofixation electrophoresis, native top-down mass spectrometry, and middle-down de novo sequencing

OBJECTIVES: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. METHODS: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. RESULTS: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin. CONCLUSIONS: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities

Combination protein biomarkers predict multiple sclerosis diagnosis and outcomes

Establishing biomarkers to predict multiple sclerosis diagnosis and prognosis has been challenging using a single biomarker approach. We hypothesised that a combination of biomarkers would increase the accuracy of prediction models to differentiate multiple sclerosis from other neurological disorders and enhance prognostication for people with multiple sclerosis. We measured 24 fluid biomarkers in the blood and cerebrospinal fluid of 77 people with multiple sclerosis and 80 people with other neurological disorders, using ELISA or Single Molecule Array assays. Primary outcomes were multiple sclerosis versus any other diagnosis, time to first relapse, and time to disability milestone (Expanded Disability Status Scale 6), adjusted for age and sex. Multivariate prediction models were calculated using the area under the curve value for diagnostic prediction, and concordance statistics (the percentage of each pair of events that are correctly ordered in time for each of the Cox regression models) for prognostic predictions. Predictions using combinations of biomarkers were considerably better than single biomarker predictions. The combination of cerebrospinal fluid [chitinase-3-like-1 + TNF-receptor-1 + CD27] and serum [osteopontin + MCP-1] had an area under the curve of 0.97 for diagnosis of multiple sclerosis, compared to the best discriminative single marker in blood (osteopontin: area under the curve 0.84) and in cerebrospinal fluid (chitinase-3-like-1 area under the curve 0.84). Prediction for time to next relapse was optimal with a combination of cerebrospinal fluid[vitamin D binding protein + Factor I + C1inhibitor] + serum[Factor B + Interleukin-4 + C1inhibitor] (concordance 0.80), and time to Expanded Disability Status Scale 6 with cerebrospinal fluid [C9 + Neurofilament-light] + serum[chitinase-3-like-1 + CCL27 + vitamin D binding protein + C1inhibitor] (concordance 0.98). A combination of fluid biomarkers has a higher accuracy to differentiate multiple sclerosis from other neurological disorders and significantly improved the prediction of the development of sustained disability in multiple sclerosis. Serum models rivalled those of cerebrospinal fluid, holding promise for a non-invasive approach. The utility of our biomarker models can only be established by robust validation in different and varied cohorts

Interrater reliability of the mind map assessment rubric in a cohort of medical students

<p>Abstract</p> <p>Background</p> <p>Learning strategies are thinking tools that students can use to actively acquire information. Examples of learning strategies include mnemonics, charts, and maps. One strategy that may help students master the tsunami of information presented in medical school is the mind map learning strategy. Currently, there is no valid and reliable rubric to grade mind maps and this may contribute to their underutilization in medicine. Because concept maps and mind maps engage learners similarly at a metacognitive level, a valid and reliable concept map assessment scoring system was adapted to form the mind map assessment rubric (MMAR). The MMAR can assess mind map depth based upon concept-links, cross-links, hierarchies, examples, pictures, and colors. The purpose of this study was to examine interrater reliability of the MMAR.</p> <p>Methods</p> <p>This exploratory study was conducted at a US medical school as part of a larger investigation on learning strategies. Sixty-six (<it>N </it>= 66) first-year medical students were given a 394-word text passage followed by a 30-minute presentation on mind mapping. After the presentation, subjects were again given the text passage and instructed to create mind maps based upon the passage. The mind maps were collected and independently scored using the MMAR by 3 examiners. Interrater reliability was measured using the intraclass correlation coefficient (<it>ICC</it>) statistic. Statistics were calculated using SPSS version 12.0 (Chicago, IL).</p> <p>Results</p> <p>Analysis of the mind maps revealed the following: concept-links <it>ICC </it>= .05 (95% CI, -.42 to .38), cross-links <it>ICC </it>= .58 (95% CI, .37 to .73), hierarchies <it>ICC </it>= .23 (95% CI, -.15 to .50), examples <it>ICC </it>= .53 (95% CI, .29 to .69), pictures <it>ICC </it>= .86 (95% CI, .79 to .91), colors <it>ICC </it>= .73 (95% CI, .59 to .82), and total score <it>ICC </it>= .86 (95% CI, .79 to .91).</p> <p>Conclusion</p> <p>The high <it>ICC </it>value for total mind map score indicates strong MMAR interrater reliability. Pictures and colors demonstrated moderate to strong interrater reliability. We conclude that the MMAR may be a valid and reliable tool to assess mind maps in medicine. However, further research on the validity and reliability of the MMAR is necessary.</p

Self-reported arm and shoulder problems in breast cancer survivors in Sub-Saharan Africa: the African Breast Cancer-Disparities in Outcomes cohort study

BACKGROUND: Arm and shoulder problems (ASP), including lymphedema, were common among women with breast cancer in high-income countries before sentinel lymph node biopsy became the standard of care. Although ASP impair quality of life, as they affect daily life activities, their frequency and determinants in Sub-Saharan Africa remain unclear. METHODS: All women newly diagnosed with breast cancer at the Namibian, Ugandan, Nigerian, and Zambian sites of the African Breast Cancer-Disparities in Outcomes (ABC-DO) cohort study were included. At each 3-month follow-up interview, women answered the EORTC-QLQ-Br23 questionnaire, including three ASP items: shoulder/arm pain, arm stiffness, and arm/hand swelling. We estimated the cumulative incidence of first self-reported ASP, overall and stratified by study and treatment status, with deaths treated as competing events. To identify determinants of ASP, we estimated cause-specific hazard ratios using Cox models stratified by study site. RESULTS: Among 1476 women, up to 4 years after diagnosis, 43% (95% CI 40-46), 36% (33-38) and 23% (20-25), respectively, self-reported having experienced arm/shoulder pain, stiffness and arm/hand swelling at least once. Although risks of self-reported ASP differed between sites, a more advanced breast cancer stage at diagnosis, having a lower socioeconomic position and receiving treatment increased the risk of reporting an ASP. CONCLUSION: ASP are very common in breast cancer survivors in Sub-Saharan Africa. They are influenced by different factors than those observed in high-income countries. There is a need to raise awareness and improve management of ASP within the African setting