Extraction, Enrichment, Solubilization, and Digestion Techniques for Membrane Proteomics

The importance of membrane proteins in biological systems is indisputable; however, their amphipathic nature makes them difficult to analyze. In this study, the most popular techniques for extraction, enrichment, solubilization, and digestion are compared, resulting in an overall improved workflow for the insoluble portion of Saccharomyces cerevisiae cell lysate. Yeast cells were successfully lysed using a French press pressure cell at 20 000 psi, and resulting proteins were fractionated prior to digestion to reduce sample complexity. The proteins were best solubilized with the addition of ionic detergent sodium deoxycholate (1%) and through the application of high-frequency sonication prior to a tryptic digestion at 37 °C. Overall, the improved membrane proteomic workflow resulted in a 26% increase in membrane protein identifications for baker's yeast. In addition, more membrane protein identifications were unique to the improved protocol. When comparing membrane proteins that were identified in the improved protocol and the standard operating procedure (176 proteins), 93% of these proteins were present in greater abundance (higher intensity) when using the improved method

The Quest for the Holy Grail: Too Many ERM Systems Are Not Enough!

Combining punctual statistical data compilation, access to real-time order and payment information, and harmonious workflow and reporting tools in one place has long been the Holy Grail for libraries seeking a reliable means for tracking costly electronic resources. This is the tale of two academic libraries that have adopted very different types of electronic resource management systems (ERMS) to attain these goals. This proceeding will provide complementary case studies of the implementation process at Binghamton University where two commercial ERM systems are used, and at The University of Texas at Tyler where an open source ERM is utilized

Insulin Status and Vascular Responses to Weight Loss in Obesity

ObjectivesThe aim of this study was to determine whether the effects of weight loss on arterial function are differentially modified by insulin status.BackgroundClinical studies suggest that plasma insulin levels may predict the extent of cardiovascular benefit achieved with weight loss in obese individuals, but mechanisms are currently unknown.MethodsWe prospectively followed 208 overweight or obese patients (body mass index [BMI] ≥25 kg/m2) receiving medical/dietary (48%) or bariatric surgical (52%) weight-loss treatment during a median period of 11.7 months (interquartile range: 4.6 to 13 months). We measured plasma metabolic parameters and vascular endothelial function using ultrasound at baseline and following weight-loss intervention and stratified analyses by median plasma insulin levels.ResultsPatients age 45 ± 1 years, with BMI 45 ± 9 kg/m2, experienced 14 ± 14% weight loss during the study period. In individuals with higher baseline plasma insulin levels (above median >12 μIU/ml; n = 99), ≥10% weight loss (compared with <10%) significantly improved brachial artery macrovascular flow-mediated vasodilation and microvascular reactive hyperemia (p < 0.05 for all). By contrast, vascular function did not change significantly in the lower insulin group (≤12 μIU/ml; n = 109) despite a similar degree of weight loss. In analyses using a 5% weight loss cut point, only microvascular responses improved in the higher insulin group (p = 0.02).ConclusionsInsulin status is an important determinant of the positive effect of weight reduction on vascular function with hyperinsulinemic patients deriving the greatest benefit. Integrated improvement in both microvascular and macrovascular function was associated with ≥10% weight loss. Reversal of insulin resistance and endothelial dysfunction may represent key therapeutic targets for cardiovascular risk reduction in obesity

Genomic analysis of focal nodular hyperplasia with associated hepatocellular carcinoma unveils its malignant potential: a case report.

Background Focal nodular hyperplasia (FNH) is typically considered a benign tumor of the liver without malignant potential. The co-occurrence of FNH and hepatocellular carcinoma (HCC) has been reported in rare cases. In this study we sought to investigate the clonal relationship between these lesions in a patient with FNH-HCC co-occurrence. Methods A 74-year-old female patient underwent liver tumor resection. The resected nodule was subjected to histologic analyses using hematoxylin and eosin stain and immunohistochemistry. DNA extracted from microdissected FNH and HCC regions was subjected to whole exome sequencing. Clonality analysis were performed using PyClone. Results Histologic analysis reveals that the nodule consists of an FNH and two adjoining HCC components with distinct histopathological features. Immunophenotypic characterization and genomic analyses suggest that the FNH is clonally related to the HCC components, and is composed of multiple clones at diagnosis, that are likely to have progressed to HCC through clonal selection and/or the acquisition of additional genetic events. Conclusion To the best of our knowledge, our work is the first study showing a clonal relationship between FNH and HCC. We show that FNH may possess the capability to undergo malignant transformation and to progress to HCC in very rare cases

1992 Blueberry Research Progress Reports

The 1992 Blueberry Research Progress Reports pertain to and report on research conducted in 1991, and were prepared for the Maine Wild Blueberry Commission and the University of Maine Wild Blueberry Advisory Committee by researchers at the University of Maine, Orono. Projects in this report include: 1992 CSRS Progress Reports: 1. Investigation of Groundwater Resources 2. Sprinkler Irrigation 3. Investigation of Preprocess Changes Leading to Berry Spoilage 4. Effect of Fertilization and Irrigation on Blueberry Quality 5. Effects of Calcium Salts and Citric Acid on Quality of Canned Lowbush Blueberries 6. Pollination of Lowbush Blueberry by Native Bees 7. Application of Heat for Controlling Insects 8. Investigations of Lowbush Blueberry Fruit Bud Cold-Hardiness 9. Steam Sterilization in Lowbush Blueberry Fields 10. Heat-Tolerant Molds 11. Vacuum Sanitation for Disease Control 12. Evaluation of Infrared Burner for Weed Control 13. Evaluation and Modification of Commercial Herbicide Wipers 14. Evaluation of Remote Sensing to Estimate Plant Cover in Lowbush Blueberry Fields 15. Comparison of Three Mechanical Blueberry Harvesters vs. Hand Raking Advisory Committee Research Reports: 16. Biology and action thresholds of secondary blueberry insects 17. Control of secondary blueberry pests 18. Control of blueberry maggot 19. Effects of calcium salts and citric acid on the quality of canned lowbush blueberries 20. The effects of postharvest handling on the dietary fiber and ellagic acid content of lowbush blueberries 21. Investigation of preprocessing changes that could lead to development of simple and inexpensive method to measure preprocessing berry spoilage 22. Determination of pesticide residue levels in fresh and processed lowbush blueberries 23. Vacuum sanitation for disease control 24. Heat-tolerant molds 25. Seedling pruning study 26. Effect of time and rate of application of Clopyralid for control of Vetch in lowbush blueberries 27. Evaluation and modification of commercial herbicide wipers 28. Effect of time of application and formulation of Hexazinone (Velpar) on Blueberry and Bunchberry 29. Evaluation of postemergence applications of Tribenuron Methyl for Bunchberry control 30. Thresholds of Dogbane and Bracken Fern by mechanical and chemical control in lowbush blueberry fields 31. Evaluation of the suitability of remote sensing to evaluate plant cover in lowbush blueberry fields 32. Evalution of infrared burner for weed control 33. Effect of time of fall pruning on growth and productivity of blueberry and evaluation of infrared burner to prune blueberries 34. Effect of Boron on lowbush blueberry fruit set and yield 35. Winter injury protection by potassium 36. Multiple cropping of wild stands 37. Nitrogen-Phosphorus study 38. Phosphorus dose/response curve 39. Investigations of lowbush blueberry fruit bud cold-hardines

Lymphatic function is required prenatally for lung inflation at birth

Mammals must inflate their lungs and breathe within minutes of birth to survive. A key regulator of neonatal lung inflation is pulmonary surfactant, a lipoprotein complex which increases lung compliance by reducing alveolar surface tension (Morgan, 1971). Whether other developmental processes also alter lung mechanics in preparation for birth is unknown. We identify prenatal lymphatic function as an unexpected requirement for neonatal lung inflation and respiration. Mice lacking lymphatic vessels, due either to loss of the lymphangiogenic factor CCBE1 or VEGFR3 function, appear cyanotic and die shortly after birth due to failure of lung inflation. Failure of lung inflation is not due to reduced surfactant levels or altered development of the lung but is associated with an elevated wet/dry ratio consistent with edema. Embryonic studies reveal active lymphatic function in the late gestation lung, and significantly reduced total lung compliance in late gestation embryos that lack lymphatics. These findings reveal that lymphatic vascular function plays a previously unrecognized mechanical role in the developing lung that prepares it for inflation at birth. They explain respiratory failure in infants with congenital pulmonary lymphangiectasia, and suggest that inadequate late gestation lymphatic function may also contribute to respiratory failure in premature infants

An international laboratory comparison of dissolved organic matter composition by high resolution mass spectrometry: Are we getting the same answer?

High-resolution mass spectrometry (HRMS) has become a vital tool for dissolved organic matter (DOM) characterization. The upward trend in HRMS analysis of DOM presents challenges in data comparison and interpretation among laboratories operating instruments with differing performance and user operating conditions. It is therefore essential that the community establishes metric ranges and compositional trends for data comparison with reference samples so that data can be robustly compared among research groups. To this end, four identically prepared DOM samples were each measured by 16 laboratories, using 17 commercially purchased instruments, using positive-ion and negative-ion mode electrospray ionization (ESI) HRMS analyses. The instruments identified ~1000 common ions in both negative- and positive-ion modes over a wide range of m/z values and chemical space, as determined by van Krevelen diagrams. Calculated metrics of abundance-weighted average indices (H/C, O/C, aromaticity, and m/z) of the commonly detected ions showed that hydrogen saturation and aromaticity were consistent for each reference sample across the instruments, while average mass and oxygenation were more affected by differences in instrument type and settings. In this paper we present 32 metric values for future benchmarking. The metric values were obtained for the four different parameters from four samples in two ionization modes and can be used in future work to evaluate the performance of HRMS instruments

The Small Molecule Inhibitor QLT0267 Radiosensitizes Squamous Cell Carcinoma Cells of the Head and Neck

BACKGROUND: The constant increase of cancer cell resistance to radio- and chemotherapy hampers improvement of patient survival and requires novel targeting approaches. Integrin-Linked Kinase (ILK) has been postulated as potent druggable cancer target. On the basis of our previous findings clearly showing that ILK transduces antisurvival signals in cells exposed to ionizing radiation, this study evaluated the impact of the small molecule inhibitor QLT0267, reported as putative ILK inhibitor, on the cellular radiation survival response of human head and neck squamous cell carcinoma cells (hHNSCC). METHODOLOGY/PRINCIPAL FINDINGS: Parental FaDu cells and FaDu cells stably transfected with a constitutively active ILK mutant (FaDu-IH) or empty vectors, UTSCC45 cells, ILK(floxed/floxed(fl/fl)) and ILK(-/-) mouse fibroblasts were used. Cells grew either two-dimensionally (2D) on or three-dimensionally (3D) in laminin-rich extracellular matrix. Cells were treated with QLT0267 alone or in combination with irradiation (X-rays, 0-6 Gy single dose). ILK knockdown was achieved by small interfering RNA transfection. ILK kinase activity, clonogenic survival, number of residual DNA double strand breaks (rDSB; gammaH2AX/53BP1 foci assay), cell cycle distribution, protein expression and phosphorylation (e.g. Akt, p44/42 mitogen-activated protein kinase (MAPK)) were measured. Data on ILK kinase activity and phosphorylation of Akt and p44/42 MAPK revealed a broad inhibitory spectrum of QLT0267 without specificity for ILK. QLT0267 significantly reduced basal cell survival and enhanced the radiosensitivity of FaDu and UTSCC45 cells in a time- and concentration-dependent manner. QLT0267 exerted differential, cell culture model-dependent effects with regard to radiogenic rDSB and accumulation of cells in the G2 cell cycle phase. Relative to corresponding controls, FaDu-IH and ILK(fl/fl) fibroblasts showed enhanced radiosensitivity, which failed to be antagonized by QLT0267. A knockdown of ILK revealed no change in clonogenic survival of the tested cell lines as compared to controls. CONCLUSIONS/SIGNIFICANCE: Our data clearly show that the small molecule inhibitor QLT0267 has potent cytotoxic and radiosensitizing capability in hHNSCC cells. However, QLT0267 is not specific for ILK. Further in vitro and in vivo studies are necessary to clarify the potential of QLT0267 as a targeted therapeutic in the clinic

Mesophilic and Thermophilic Conditions Select for Unique but Highly Parallel Microbial Communities to Perform Carboxylate Platform Biomass Conversion

The carboxylate platform is a flexible, cost-effective means of converting lignocellulosic materials into chemicals and liquid fuels. Although the platform's chemistry and engineering are well studied, relatively little is known about the mixed microbial communities underlying its conversion processes. In this study, we examined the metagenomes of two actively fermenting platform communities incubated under contrasting temperature conditions (mesophilic 40°C; thermophilic 55°C), but utilizing the same inoculum and lignocellulosic feedstock. Community composition segregated by temperature. The thermophilic community harbored genes affiliated with Clostridia, Bacilli, and a Thermoanaerobacterium sp, whereas the mesophilic community metagenome was composed of genes affiliated with other Clostridia and Bacilli, Bacteriodia, γ-Proteobacteria, and Actinobacteria. Although both communities were able to metabolize cellulosic materials and shared many core functions, significant differences were detected with respect to the abundances of multiple Pfams, COGs, and enzyme families. The mesophilic metagenome was enriched in genes related to the degradation of arabinose and other hemicellulose-derived oligosaccharides, and the production of valerate and caproate. In contrast, the thermophilic community was enriched in genes related to the uptake of cellobiose and the transfer of genetic material. Functions assigned to taxonomic bins indicated that multiple community members at either temperature had the potential to degrade cellulose, cellobiose, or xylose and produce acetate, ethanol, and propionate. The results of this study suggest that both metabolic flexibility and functional redundancy contribute to the platform's ability to process lignocellulosic substrates and are likely to provide a degree of stability to the platform's fermentation processes