The design, development and construction of a low cost multi-purpose computer/interface system for on-line experimentation: applications to time averaging of NMR spectra and other chemical instrumentation problems

The project goal was the development of a low cost multipurpose microprocessor interfacing system that would be adaptable to a number of commonly encountered chemical instruments (such as NRM, GC, IR, and UV-VIS.) for the purpose of control, data acquisition processing. The Altair Microprocessor with 4K random access memory was constructed from kit form; it functions as the computer portion of the system. The digital to analog and analog to digital signal conversion elements as well as timing, control and other necessary logic functions are incorporated in the electronic interface portion of the system. The computer, interface, principles of time averaging, and the philosophy of interfacing in general, are the subjects of this report. The Perkin Elmer R-24A NMR spectrometer is presently attached to the computer/ interface system so that signal time averaging on low concentration samples may be accomplished. A by-product of this particular system is a library of spectra stored on binary coded paper tape which can be used to generate spectra without having to make up samples. Future applications of the system are also considered in this thesis

Mass Spectrometry-based Methods for Phosphorylation Site Mapping of Hyperphosphorylated Proteins Applied to Net1, a Regulator of Exit from Mitosis in Yeast

Prior to anaphase in Saccharomyces cerevisiae, Cdc14 protein phosphatase is sequestered within the nucleolus and inhibited by Net1, a component of the RENT complex in budding yeast. During anaphase the RENT complex disassembles, allowing Cdc14 to migrate to the nucleus and cytoplasm where it catalyzes exit from mitosis. The mechanism of Cdc14 release appears to involve the polo-like kinase Cdc5, which is capable of promoting the dissociation of a recombinant Net1·Cdc14 complex in vitro by phosphorylation of Net1. We report here the phosphorylation site mapping of recombinant Net1 (Net1N) and a mutant Net1N allele (Net1N-19m) with 19 serines or threonines mutated to alanine. A variety of chromatographic and mass spectrometric-based strategies were used, including immobilized metal-affinity chromatography, alkaline phosphatase treatment, matrix-assisted laser-desorption post-source decay, and a multidimensional electrospray mass spectrometry-based approach. No one approach was able to identify all phosphopeptides in the tryptic digests of these proteins. Most notably, the presence of a basic residue near the phosphorylated residue significantly hampered the ability of alkaline phosphatase to hydrolyze the phosphate moiety. A major goal of research in proteomics is to identify all proteins and their interactions and post-translational modification states. The failure of any single method to identify all sites in highly phosphorylated Net1N, however, raises significant concerns about how feasible it is to map phosphorylation sites throughout the proteome using existing technologies

Extracellular matrix signatures of human primary metastatic colon cancers and their metastases to liver

Background: Colorectal cancer is the third most frequently diagnosed cancer and the third cause of cancer deaths in the United States. Despite the fact that tumor cell-intrinsic mechanisms controlling colorectal carcinogenesis have been identified, novel prognostic and diagnostic tools as well as novel therapeutic strategies are still needed to monitor and target colon cancer progression. We and others have previously shown, using mouse models, that the extracellular matrix (ECM), a major component of the tumor microenvironment, is an important contributor to tumor progression. In order to identify candidate biomarkers, we sought to define ECM signatures of metastatic colorectal cancers and their metastases to the liver. Methods: We have used enrichment of extracellular matrix (ECM) from human patient samples and proteomics to define the ECM composition of primary colon carcinomas and their metastases to liver in comparison with normal colon and liver samples. Results: We show that robust signatures of ECM proteins characteristic of each tissue, normal and malignant, can be defined using relatively small samples from small numbers of patients. Comparisons with gene expression data from larger cohorts of patients confirm the association of subsets of the proteins identified by proteomic analysis with tumor progression and metastasis. Conclusions: The ECM protein signatures of metastatic primary colon carcinomas and metastases to liver defined in this study, offer promise for development of diagnostic and prognostic signatures of metastatic potential of colon tumors. The ECM proteins defined here represent candidate serological or tissue biomarkers and potential targets for imaging of occult metastases and residual or recurrent tumors and conceivably for therapies. Furthermore, the methods described here can be applied to other tumor types and can be used to investigate other questions such as the role of ECM in resistance to therapy

Pulmonary artery stiffness in chronic obstructive pulmonary disease (copd) and emphysema: The Multi-Ethnic Study of Atherosclerosis (MESA) COPD Study

Purpose: Chronic obstructive pulmonary disease (COPD) and particularly emphysema are characterized by stiffness of the aorta, due in part to accelerated elastin degradation in the lungs and aorta. Stiffness of the pulmonary arteries (PAs) may also be increased in COPD and emphysema, but data are lacking. We assessed PA stiffness using MRI in patients with COPD and related these measurements to COPD severity and percent emphysema. Materials and Methods: The Multi-Ethnic Study of Atherosclerosis (MESA) COPD Study recruited 290 participants, age 50–79 years with 10 or more packyears and free of clinical cardiovascular disease. COPD severity were defined on postbronchodilator spirometry by ATS/ERS criteria. Percent emphysema was defined as the percentage of regions of the lung < -950 Hounsfield units on full-lung computed tomography (CT). PA stain was defined by the percent change in cross-sectional PA area between systole and diastole on MRI. Blood flow across the tricuspid and mitral valves was assessed by phase-contrast MRI for determination of the ventricular diastolic dysfunction (E/A ratio). Results: PA strain was reduced in COPD compared with controls (P = 0.002) and was inversely correlated with COPD severity (P = 0.004). PA strain was inversely associated to percent emphysema (P = 0.01). PA strain was also markedly correlated with right ventricular diastolic dysfunction measured by E/A ratios in the fully adjusted mix models (P = 0.02). Conclusion: PA strain is reduced in COPD, related in part to percent emphysema on CT scan, which may have implications for pulmonary small vessel flow and right ventricular function. Level of Evidence: 2 Technical Efficacy: Stage

Workshop on Mars Sample Return Science

Martian magnetic history; quarantine issues; surface modifying processes; climate and atmosphere; sampling sites and strategies; and life sciences were among the topics discussed

Single Sessions of High-Definition Transcranial Direct Current Stimulation Do Not Alter Lower Extremity Biomechanical or Corticomotor Response Variables Post-stroke

Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique used to modulate cortical activity. However, measured effects on clinically relevant assessments have been inconsistent, possibly due to the non-focal dispersion of current from traditional two electrode configurations. High-definition (HD)-tDCS uses a small array of electrodes (N = 5) to improve targeted current delivery. The purpose of this study was to determine the effects of a single session of anodal and cathodal HD-tDCS on gait kinematics and kinetics and the corticomotor response to transcranial magnetic stimulation (TMS) in individuals post-stroke. We hypothesized that ipsilesional anodal stimulation would increase the corticomotor response to TMS leading to beneficial changes in gait. Eighteen participants post-stroke (average age: 64.8 years, SD: 12.5; average months post-stroke: 54, SD: 42; average lower extremity Fugl-Meyer score: 26, SD: 6) underwent biomechanical and corticomotor response testing on three separate occasions prior to and after HD-tDCS stimulation. In a randomized order, anodal, cathodal, and sham HD-tDCS were applied to the ipsilesional motor cortex for 20 min while participants pedaled on a recumbent cycle ergometer. Gait kinetic and kinematic data were collected while walking on an instrumented split-belt treadmill with motion capture. The corticomotor response of the paretic and non-paretic tibialis anterior (TA) muscles were measured using neuronavigated TMS. Repeated measures ANOVAs using within-subject factors of time point (pre, post) and stimulation type (sham, anodal, cathodal) were used to compare effects of HD-tDCS stimulation on measured variables. HD-tDCS had no effect on over ground walking speed (P > 0.41), or kinematic variables (P > 0.54). The corticomotor responses of the TA muscles were also unaffected by HD-tDCS (resting motor threshold, P = 0.15; motor evoked potential (MEP) amplitude, P = 0.25; MEP normalized latency, P = 0.66). A single session of anodal or cathodal HD-tDCS delivered to a standardized ipsilesional area of the motor cortex does not appear to alter gait kinematics or corticomotor response post-stroke. Repeated sessions and individualized delivery of HD-tDCS may be required to induce beneficial plastic effects. Contralesional stimulation should also be investigated due to the altered interactions between the cerebral hemispheres post-stroke

Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ΔmycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ΔmycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems

Spatial Analysis of Arabidopsis thaliana Gene Expression in Response to Turnip mosaic virus Infection

Virus-infected leaf tissues comprise a heterogeneous mixture of cells at different stages of infection. The spatial and temporal relationships between sites of virus accumulation and the accompanying host responses, such as altered host gene expression, are not well defined. To address this issue, we utilized Turnip mosaic virus (TuMV) tagged with the green fluorescent protein to guide the dissection of infection foci into four distinct zones. The abundance of Arabidopsis thaliana mRNA transcripts in each of the four zones then was assayed using the Arabidopsis ATH1 GeneChip oligonucleotide microarray (Affymetrix). mRNA transcripts with significantly altered expression profiles were determined across gradients of virus accumulation spanning groups of cells in and around foci at different stages of infection. The extent to which TuMV-responsive genes were up- or downregulated primarily correlated with the amount of virus accumulation regardless of gene function. The spatial analysis also allowed new suites of coordinately regulated genes to be identified that are associated with chloroplast functions (decreased), sulfate assimilation (decreased), cell wall extensibility (decreased), and protein synthesis and turnover (induced). The functions of these downregulated genes are consistent with viral symptoms, such as chlorosis and stunted growth, providing new insight into mechanisms of pathogenesis

Climate vulnerability assessment for Pacific salmon and steelhead in the California Current Large Marine Ecosystem.

Major ecological realignments are already occurring in response to climate change. To be successful, conservation strategies now need to account for geographical patterns in traits sensitive to climate change, as well as climate threats to species-level diversity. As part of an effort to provide such information, we conducted a climate vulnerability assessment that included all anadromous Pacific salmon and steelhead (Oncorhynchus spp.) population units listed under the U.S. Endangered Species Act. Using an expert-based scoring system, we ranked 20 attributes for the 28 listed units and 5 additional units. Attributes captured biological sensitivity, or the strength of linkages between each listing unit and the present climate; climate exposure, or the magnitude of projected change in local environmental conditions; and adaptive capacity, or the ability to modify phenotypes to cope with new climatic conditions. Each listing unit was then assigned one of four vulnerability categories. Units ranked most vulnerable overall were Chinook (O. tshawytscha) in the California Central Valley, coho (O. kisutch) in California and southern Oregon, sockeye (O. nerka) in the Snake River Basin, and spring-run Chinook in the interior Columbia and Willamette River Basins. We identified units with similar vulnerability profiles using a hierarchical cluster analysis. Life history characteristics, especially freshwater and estuary residence times, interplayed with gradations in exposure from south to north and from coastal to interior regions to generate landscape-level patterns within each species. Nearly all listing units faced high exposures to projected increases in stream temperature, sea surface temperature, and ocean acidification, but other aspects of exposure peaked in particular regions. Anthropogenic factors, especially migration barriers, habitat degradation, and hatchery influence, have reduced the adaptive capacity of most steelhead and salmon populations. Enhancing adaptive capacity is essential to mitigate for the increasing threat of climate change. Collectively, these results provide a framework to support recovery planning that considers climate impacts on the majority of West Coast anadromous salmonids