BIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF MURINE-TUMOR SPECIFIC TRANSPLANTATION ANTIGENS (ISOTACHOPHORESIS, IMMUNOGENICITY)

Tumor-specific transplantation antigens (TSTA) are individually distinct neoantigens expressed on the cells of chemically-induced neoplasms. TSTA are operationally defined by immunization of syngeneic mice against challenge with viable tumor cells. Immunization with cell surface or extracted TSTA induces specific resistance to transplanted tumor cells. The biological and biochemical nature of TSTA was investigated in the 3-methylcholanthrene-induced fibrosarcomas of female C3H/HeJ mice, MCA-F and MCA-D. Tumor cell suspensions were extracted by treatment with 3M KCl or 2.5% butanol solutions and the TSTA was partially purified by preparative isoelectric focusing. The isoelectric pH of TSTA purified from 3M KCl extracts was 5.8-6.0, and from butanol extracts was 6.4-6.6. Whereas immunization with 10('5) and 10('6) irradiated tumor cells induces complete rejection of tumor cell challenge over a two-fold-log dose range, immunization with ug quantities within a one-fold-log dose range of extracted TSTA induces only partial resistance to tumor challenge. Reduced immunogenicity of extracted TSTA is hypothesized to result from immunization of mice with insufficiently purified TSTA preparations. The hypothesis predicts that immunization with highly purified TSTA, free from interfering substances, induces complete rejection of tumor challenge over a broad dose range. To test the hypothesis preparative isotachophoresis (pITP) was used to purify TSTA from electrofocused TSTA fractions. Significant purification was achieved, as immunization with 15 pg to 1.5 ug (5 logs) of pITP-purified TSTA extracted from the MCA-F, or with 1 pg to 10 ng (4 logs) of TSTA from the MCA-D tumor induced specific resistance to tumor challenge. Despite 50,000 fold purification of TSTA, immunization induced partial, not complete, rejection of transplanted tumor cells. This suggests a clear dissociation of the immunogenicity and purification of extracted TSTA, indicating that the induction of partial immunity to tumor challenge is an intrinsic property of extracted TSTA.^

Bentgrass response to K fertilization and K release rates from eight sand rootzone sources used in putting green construction.

There is a lack of plant response to fertilizer K in some sandy soils even though routine soil tests for soil available K are shown to be low. This lack of plant response to K fertilizer application may be explained by K release from nonexchangeable forms. Greenhouse and laboratory experiments were conducted to evaluate (a) response of bentgrass (Agrostis palustris [Agrostis stolonifera var. palustris]) cv. Pencross grown in rootzones with different sand sources to K fertilizer application and (b) K release from nonexchangeable forms from the different sand sources as an index to K availability. Experimental variables in the greenhouse were 2 K levels (0 and 250 mg K/kg soil) and 8 sand rootzone sources. Rootzone soils were sub-irrigated to ensure no K loss from leaching. Two laboratory methods (boiling 1 M HNO3 extraction and continuous leaching with 0.01 M HCl) and total K uptake by the bentgrass were employed to index K release from nonexchangeable forms for each rootzone source. K fertilizer application significantly increased bentgrass yield growing in one rootzone source and root weight in 3 rootzone sources. K uptake by bentgrass and the 2 laboratory methods showed important differences in K release from the sand rootzones. The K removed by the 2 laboratory methods was closely related to leaf tissue K and K uptake, with the 1 M HNO3 extraction method providing the closest fit. The release of K from primary minerals in some rootzones with high sand content is proceeding at rates to satisfy bentgrass requirements for K. The 1 M HNO3 extraction method may provide an alternative to the routine laboratory procedures presently being used to measure the extractable K in sand-based constructed putting greens by measuring K contributed by nonexchangeable forms.

Cataract surgery in cancer patients and its impact on quality of life as measured by the National Eye Institute Visual Functioning Questionnaire - 25 (NEI-VFQ-25)

Purpose. The measurement of quality of life has become an important topic in healthcare and in the allocation of limited healthcare resources. Improving the quality of life (QOL) in cancer patients is paramount. Cataract removal and lens implantation appears to improve patient well-being of cancer patients, though a formal measurement has never been published in the US literature. In this current study, National Eye Institute Visual Functioning Questionnaire (NEI-VFQ-25), a validated vision quality of life metric, was used to study the change in vision-related quality of life in cancer patients who underwent cataract extraction with intraocular lens implantation. ^ Methods. Under an IRB approved protocol, cancer patients who underwent cataract surgery with intraocular lens implantation (by a single surgeon) from December 2008 to March 2011, and who had completed a pre- and postoperative NEI-VFQ-25 were retrospectively reviewed. Post-operative data was collected at their routine 4-6 week post-op visit. Patients' demographics, cancer history, their pre and postoperative ocular examinations, visual acuities, and NEI-VFQ-25 with twelve components were included in the evaluation. The responses were evaluated using the Student t test, Spearman correlation and Wilcoxon signed rank test. ^ Results. 63 cases of cataract surgery (from 54 patients) from the MD Anderson Cancer Center were included in the study. Cancer patients had a significant improvement in the visual acuity (P<0.0001) postoperatively, along with a significant increase in vision-related quality of life (P<0.0001). Patients also had a statistically significant improvement in ten of the twelve subcategories which are addressed in the NEI-VFQ-25. ^ Conclusions. In our study, cataract extraction and intraocular implantation showed a significant impact on the vision-related quality of life in cancer patients. Although this study includes a small sample size, it serves as a positive pilot study to evaluate and quantify the impact of a surgical intervention on QOL in cancer patients and may help to design a larger study to measure vision related QOL per dollar spent for health care cost in cancer patients.^

Radiomics of NSCLC: Quantitative CT Image Feature Characterization and Tumor Shrinkage Prediction

Radiomics is the high-throughput extraction and analysis of quantitative image features. For non-small cell lung cancer (NSCLC) patients, radiomics can be applied to standard of care computed tomography (CT) images to improve tumor diagnosis, staging, and response assessment. The first objective of this work was to show that CT image features extracted from pre-treatment NSCLC tumors could be used to predict tumor shrinkage in response to therapy. This is important since tumor shrinkage is an important cancer treatment endpoint that is correlated with probability of disease progression and overall survival. Accurate prediction of tumor shrinkage could also lead to individually customized treatment plans. To accomplish this objective, 64 stage NSCLC patients with similar treatments were all imaged using the same CT scanner and protocol. Quantitative image features were extracted and principal component regression with simulated annealing subset selection was used to predict shrinkage. Cross validation and permutation tests were used to validate the results. The optimal model gave a strong correlation between the observed and predicted shrinkages with . The second objective of this work was to identify sets of NSCLC CT image features that are reproducible, non-redundant, and informative across multiple machines. Feature sets with these qualities are needed for NSCLC radiomics models to be robust to machine variation and spurious correlation. To accomplish this objective, test-retest CT image pairs were obtained from 56 NSCLC patients imaged on three CT machines from two institutions. For each machine, quantitative image features with concordance correlation coefficient values greater than 0.90 were considered reproducible. Multi-machine reproducible feature sets were created by taking the intersection of individual machine reproducible feature sets. Redundant features were removed through hierarchical clustering. The findings showed that image feature reproducibility and redundancy depended on both the CT machine and the CT image type (average cine 4D-CT imaging vs. end-exhale cine 4D-CT imaging vs. helical inspiratory breath-hold 3D CT). For each image type, a set of cross-machine reproducible, non-redundant, and informative image features was identified. Compared to end-exhale 4D-CT and breath-hold 3D-CT, average 4D-CT derived image features showed superior multi-machine reproducibility and are the best candidates for clinical correlation.

Mechanism of subunit interaction and DNA binding of the heterotrimeric CCAAT binding factor CBF

Many eukaryotic promoters contain a CCAAT element at a site close ($-$80 to $-$120) to the transcription initiation site. CBF (CCAAT Binding Factor), also called NF-Y and CP1, was initially identified as a transcription factor binding to such sites in the promoters of the Type I collagen, albumin and MHC class II genes. CBF is a heteromeric transcription factor and purification and cloning of two of the subunits, CBF-A and CBF-B revealed that it was evolutionarily conserved with striking sequence identities with the yeast polypeptides HAP3 and HAP2, which are components of a CCAAT binding factor in yeast. Recombinant CBF-A and CBF-B however failed to bind to DNA containing CCAAT sequences. Biochemical experiments led to the identification of a third subunit, CBF-C which co-purified with CBF-A and complemented the DNA binding of recombinant CBF-A and CBF-B. We have recently isolated CBF-C cDNAs and have shown that bacterially expressed purified CBF-C binds to CCAAT containing DNA in the presence of recombinant CBF-A and CBF-B. Our experiments also show that a single molecule each of all the three subunits are present in the protein-DNA complex. Interestingly, CBF-C is also evolutionarily conserved and the conserved domain between CBF-C and its yeast homolog HAP5 is sufficient for CBF-C activity. Using GST-pulldown experiments we have demonstrated the existence of protein-protein interaction between CBF-A and CBF-C in the absence of CBF-B and DNA. CBF-B on other hand, requires both CBF-A and CBF-C to form a ternary complex which then binds to DNA. Mutational studies of CBF-A have revealed different domains of the protein which are involved in CBF-C interaction and CBF-B interaction. In addition, CBF-A harbors a domain which is involved in DNA recognition along with CBF-B. Dominant negative analogs of CBF-A have also substantiated our initial observation of assembly of CBF subunits. Our studies define a novel DNA binding structure of heterotrimeric CBF, where the three subunits of CBF follow a particular pathway of assembly of subunits that leads to CBF binding to DNA and activating transcription. ^

Proceedings of the Twenty-Sixth Annual Biochemical Engineering Symposium

This volume contains the Proceedings of the Twenty-Sixth Annual Biochemical Engineering Symposium held at Kansas State University on September 21, 1996. The program included 10 oral presentations and 14 posters. Some of the papers describe the progress of ongoing projects, and others contain the results of completed projects. Only brief summaries are given of some of the papers; many of the papers will be published in full elsewhere. A listing of those who attended is given below. ContentsForeign Protein Production from SV40 Early Promoter in Continuous Cultures of Recombinant CHO Cells - Gautam Banik, Paul Todd, and Dhinakar Kampala Enhanced Cell Recruitment Due to Cell-Cell Interactions - Brad Farlow and Matthias Nollert The Recirculation of Hybridoma Suspension Cultures: Effects on Cell Death, Metabolism and Mab Productivity - Peng Jin and Carole A. Heath The Importance of Enzyme Inactivation and Self-Recovery in Cometabolic Biodegradation of Chlorinated Solvents - Xi-Hui Zhang, Shanka Banerji, and Rakesh Bajpai Phytoremediation of VOC contaminated Groundwater using Poplar Trees - Melissa Miller, Jason Dana, L.C. Davis, Murlidharan Narayanan, and L.E. Erickson Biological Treatment of Off-Gases from Aluminum Can Production: Experimental Results and Mathematical Modeling - Adeyma Y. Arroyo, Julio Zimbron, and Kenneth F. Reardon Inertial Migration Based Separation of Chlorella Microalgae in Branched Tubes - N.M. Poflee, A.L. Rakow, D.S. Dandy, M.L. Chappell, and M.N. Pons Contribution of Electrochemical Charge to Protein Partitioning in Aqueous Two-Phase Systems - Weiyu Fan and Charles C. Glatz Biodegradation of Some Commercial Surfactants Used in Bioremediation - Jun Gu, G.W. Preckshot, S.K. Banerji, and Rakesh Bajpai Modeling the Role of Biomass in Heavy Metal Transport Ln Vadose Zone - K.V. Nedunuri, L.E. Erickson, and R.S. Govindaraju Multivariable Statistical Methods for Monitoring Process Quality: Application to Bioinsecticide Production by 73 89 Bacillus Thuringiensis - c. Puente and M.N. Karim The Use of Polymeric Flocculants in Bacterial Lysate Streams - H. Graham, A.S. Cibulskas and E.H. Dunlop Effect of Water Content on transport of Trichloroethylene in a Chamber with Alfalfa Plants - Muralidharan Narayanan, Jiang Hu, Lawrence C. Davis, and Larry E. Erickson Detection of Specific Microorganisms using the Arbitrary Primed PCR in the Bacterial Community of Vegetated Soil - X. Wu and L.C. Davis Flux Enhancement Using Backpulsing - V.T. Kuberkar and R.H. Davis Chromatographic Purification of Oligonucleotides: Comparison with Electrophoresis - Stephen P. Cape, Ching-Yuan Lee, Kevin Petrini, Sean Foree, Micheal G. Sportiello and Paul Todd Determining Singular Arc Control Policies for Bioreactor Systems Using a Modified Iterative Dynamic Programming Algorithm - Arun Tholudur and W. Fred Ramirez Pressure Effect on Subtilisins Measured via FTIR, EPR and Activity Assays, and Its Impact on Crystallizations - J.N. Webb, R.Y. Waghmare, M.G. Bindewald, T.W. Randolph, J.F. Carpenter, C.E. Glatz Intercellular Calcium Changes in Endothelial Cells Exposed to Flow - Laura Worthen and Matthias Nollert Application of Liquid-Liquid Extraction in Propionic Acid Fermentation - Zhong Gu, Bonita A. Glatz, and Charles E. Glatz Purification of Recombinant T4 Lysozyme from E. Coli: Ion-Exchange Chromatography - Weiyu Fan, Matt L. Thatcher, and Charles E. Glatz Recovery and Purification of Recombinant Beta-Glucuronidase from Transgenic Corn - Ann R. Kusnadi, Roque Evangelista, Zivko L. Nikolov, and John Howard Effects of Auxins and cytokinins on Formation of Catharanthus Roseus G. Don Multiple Shoots - Ying-Jin Yuan, Yu-Min Yang, Tsung-Ting Hu, and Jiang Hu Fate and Effect of Trichloroethylene as Nonaqueous Phase Liquid in Chambers with Alfalfa - Qizhi Zhang, Brent Goplen, Sara Vanderhoof, Lawrence c. Davis, and Larry E. Erickson Oxygen Transport and Mixing Considerations for Microcarrier Culture of Mammalian Cells in an Airlift Reactor - Sridhar Sunderam, Frederick R. Souder, and Marylee Southard Effects of Cyclic Shear Stress on Mammalian Cells under Laminar Flow Conditions: Apparatus and Methods - M.L. Rigney, M.H. Liew, and M.Z. Southard

Iron and manganese in bottom sediments and interstitial waters sampled in the White Sea on August 21-30, 2003

Iron and manganese in bottom sediments studied along the sublatitudinal transect from Kandalaksha to Arkhangelsk are characterized by various contents and speciations depending on sedimentation environment, grain size of sediments, and diagenetic processes. The latter include redistribution of reactive forms leading to enrichment in Fe and Mn of surface sediments, formation of films, incrustations, and ferromanganese nodules. Variations in total Fe content (2-8%) are accompanied by changes in concentration of its reactive forms (acid extraction) and concentration of dissolved Fe in interstitial waters (1-14 µM). Variations in Mn content in bottom sediments (0.03-3.7%) and interstitial waters (up to 500 µM) correspond to high diagenetic mobility of this element. Changes in oxidation degree of chemical elements result in redox stratification of sediment strata with maximum concentrations of Fe, Mn, and sulfides. Organic matter of bottom sediments with considerable terrestrial constituent is oxidized by bottom water oxygen mainly at the sediment surface or in anaerobic conditions within the sediment strata. The role of inorganic components in organic matter oxidation changes from surface layer bottom sediments (where manganese oxyhydroxide dominates among oxidants) to deeper layers (where sulfate of interstitial water serves as the main oxidant). Differences in river runoff and hydrodynamics are responsible for geochemical asymmetry of the transect. The deep Kandalaksha Bay serves as a sediment trap for manganese (Mn content in sediments varies within 0.5-0.7%), whereas the sedimentary environment in the Dvina Bay promotes its removal from bottom sediments (Mn 0.05%).

(Table 3) Concentrations of polybrominated diphenyl ether and congeners in blubber samples from whales, seals and porpoises between 1986-2009

A selection of PBDE congeners was analyzed in pooled blubber samples of pilot whale (Globicephala melas), ringed seal (Phoca hispida), minke whale (Balaenoptera acutorostrata), fin whale (Balaenoptera physalus), harbor porpoise (Phocoena phocoena), hooded seal (Cystophora cristata) and Atlantic white-sided dolphin (Lagenorhynchus acutus), covering a time period of more than 20 years (1986-2009). The analytes were extracted and cleaned-up using open column extraction and multi-layer silica gel column chromatography, and the analysis was performed on a GC-MS system operating in the NCI mode. The highest PBDE levels were found in the toothed whale species pilot whale and white-sided dolphin, and the lowest levels in fin whales and ringed seals. One-sided analyses of variance (ANOVA) followed by Tukey comparisons of means were applied to test for differences between years and sampling areas. Due to inter-year sampling variability, only general comparisons of PBDE concentrations between different sampling areas could be made. Differences in PBDE concentrations between three sampling periods, from 1986 to 2007, were evaluated in samples of pilot whales, ringed seals, white-sided dolphins and hooded seals. The highest PBDE levels were found in samples from the late 1990s or beginning of 2000, possibly reflecting the increase in the global production of technical PBDE mixtures in the 1990s. The levels of BDE #153 and #154 increased relative to the total PBDE concentration in some of the species in recent years, which may indicate an increased relative exposure to higher brominated congeners. In order to assess the effect of measures taken in legally binding international agreements, it is important to continuously monitor POPs such as PBDEs in sub-Arctic and Arctic environments.

Mean chemical characteristics and volcano signatures of ice cores from Belukha, the Everest and three Svalbard sites

Ice cores from outside the Greenland and Antarctic ice sheets are difficult to date because of seasonal melting and multiple sources (terrestrial, marine, biogenic and anthropogenic) of sulfates deposited onto the ice. Here we present a method of volcanic sulfate extraction that relies on fitting sulfate profiles to other ion species measured along the cores in moving windows in log space. We verify the method with a well dated section of the Belukha ice core from central Eurasia. There are excellent matches to volcanoes in the preindustrial, and clear extraction of volcanic peaks in the post-1940 period when a simple method based on calcium as a proxy for terrestrial sulfate fails due to anthropogenic sulfate deposition. We then attempt to use the same statistical scheme to locate volcanic sulfate horizons within three ice cores from Svalbard and a core from Mount Everest. Volcanic sulfate is <5% of the sulfate budget in every core, and differences in eruption signals extracted reflect the large differences in environment between western, northern and central regions of Svalbard. The Lomonosovfonna and Vestfonna cores span about the last 1000 years, with good extraction of volcanic signals, while Holtedahlfonna which extends to about AD1700 appears to lack a clear record. The Mount Everest core allows clean volcanic signal extraction and the core extends back to about AD700, slightly older than a previous flow model has suggested. The method may thus be used to extract historical volcanic records from a more diverse geographical range than hitherto.

Measurements of seawater and aerosol particles during POLARSTERN cruise ANT-XXVII/4

An analytical method for the determination of the alpha dicarbonyls glyoxal (GLY) and methylglyoxal (MGLY) from seawater and marine aerosol particles is presented. The method is based on derivatization with o-(2,3,4,5,6-Pentafluorobenzyl)-hydroxylamine (PFBHA) reagent, solvent extraction and GC-MS (SIM) analysis. The method showed good precision (RSD < 10%), sensitivity (detection limits in the low ng/l range), and accuracy (good agreement between external calibration and standard addition). The method was applied to determine GLY and MGLY in oceanic water sampled during the Polarstern cruise ANT XXVII/4 from Capetown to Bremerhaven in spring 2011. GLY and MGLY were determined in the sea surface microlayer (SML) of the ocean and corresponding bulk water (BW) with average concentrations of 228 ng/l (GLY) and 196 ng/l (MGLY). The results show a significant enrichment (factor of 4) of GLY and MGLY in the SML. Furthermore, marine aerosol particles (PM1) were sampled during the cruise and analyzed for GLY (average concentration 0.19 ng/m**3) and MGLY (average concentration 0.15 ng/m**3). On aerosol particles, both carbonyls show a very good correlation with oxalate, supporting the idea of a secondary formation of oxalic acid via GLY and MGLY. Concentrations of GLY and MGLY in seawater and on aerosol particles were correlated to environmental parameters such as global radiation, temperature, distance to the coastline and biological activity. There are slight hints for a photochemical production of GLY and MGLY in the SML (significant enrichment in the SML, higher enrichment at higher temperature). However, a clear connection of GLY and MGLY to global radiation as well as to biological activity cannot be concluded from the data. A slight correlation between GLY and MGLY in the SML and in aerosol particles could be a hint for interactions, in particular of GLY, between seawater and the atmosphere.

Non-isoprenoidal intact polar lipids, isoprenoidal and non-isoprenoidal core lipids, isoprenoidal intact polar lipids of the Black Sea site GeoB15105 sampled during Meteor cruise M84/1 in February 2011

This PhD thesis focused on the analysis and application of microbial membrane lipids as biomarkers in marine sediments. Existing protocols for lipid extraction from marine sediments and biomass were modified. In addition, recent protocols based on high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) as well as state-of-the-art mass spectrometric analysis by quadrupole time-of-flight (MSqTOF) and the triple quadrupole (MSQQQ) mass spectrometer were used to investigate matrix effects and evaluate the reliability of quantitative analysis in marine environmental samples. The improved lipid extraction and quantification were used to analyze Black Sea water column and sediments samples to a depth of 8 meters from site GeoB 15105 taken during cruise M84/1 (DARCSEAS) with R/V Meteor to apply lipid analysis in benthic bio systems. With this component specific differentiation between planktonic and benthic lipid signature we assessed possible lipid sources. Here, this high detail lipid fingerprinting allowed us to observe changes in the head group and lipid core structures of the intact polar lipids according to the geochemical zonation.

(Table 2) Polybrominated diphenyl ether concentration in blubber samples from whales, seals and porpoises between 1986-2009

A selection of MeO-BDE and BDE congeners were analyzed in pooled blubber samples of pilot whale (Globicephala melas), ringed seal (Phoca hispida), minke whale (Balaenoptera acutorostrata), fin whale (Balaenoptera physalus), harbor porpoise (Phocoena phocoena), hooded seal (Cystophora cristata), and Atlantic white-sided dolphin (Lagenorhynchus acutus), covering a time period of more than 20 years (1986-2009). The analytes were extracted and cleaned-up using open column extraction and multi-layer silica gel column chromatography. The analysis was performed using both low resolution and high resolution GC-MS. MeO-PBDE concentrations relative to total PBDE concentrations varied greatly between sampling periods and species. The highest MeO-PBDE levels were found in the toothed whale species pilot whale and white-sided dolphin, often exceeding the concentration of the most abundant PBDE, BDE-47. The lowest MeO-PBDE levels were found in fin whales and ringed seals. The main MeO-BDE congeners were 6-MeO-BDE47 and 2'-MeO-BDE68. A weak correlation only between BDE47 and its methoxylated analog 6-MeO-BDE47 was found and is indicative of a natural source for MeO-PBDEs.

Land based mesocosm experiment studying the simultaneous impact of warming and acidification on the planktonic food web of the Eastern Mediterranean

A land based mesocosm experiment focusing on the study of the simultaneous impact of warming and acidification on the planktonic food web of the Eastern Mediterranean took place in August-September 2013 at the mesocosm facilities of HCMR in Crete (CRETACOSMOS). Two different pCO2 (present day and predicted for year 2100) were applied in triplicate mesocosms of 3 m**3. This was tested in two different temperatures (ambient seawater T and ambient T plus 3°C). Twelve mesocosms in total were incubated in two large concrete tanks. Temperature was controlled by sophisticated, automated systems. A large variety of chemical, biological and biochemical variables were studied, including salinity, temperature, light and alkalinity measurements, inorganic and organic, particulate and dissolved, nutrient analyses, biological stock (Chla concentration, enumeration and community composition of microbial, phyto- and zooplankton organisms) and rate (primary, bacterial, viral production, copepod egg production, zooplankton grazing, N2 fixation, P uptake) measurements, bacterial DNA extraction and phytoplankton transcriptomics, calcifiers analyses. Twenty three scientists from 6 Institutes and 5 countries participated in this experiment.