The cystatin C/creatinine ratio, a marker of glomerular filtration quality: associated factors, reference intervals, and prediction of morbidity and mortality in healthy seniors.

The ratio of cystatin C (cysC) to creatinine (crea) is regarded as a marker of glomerular filtration quality associated with cardiovascular morbidities. We sought to determine reference intervals for serum cysC-crea ratio in seniors. Furthermore, we sought to determine whether other low-molecular weight molecules exhibit a similar behavior in individuals with altered glomerular filtration quality. Finally, we investigated associations with adverse outcomes. A total of 1382 subjectively healthy Swiss volunteers aged 60 years or older were enrolled in the study. Reference intervals were calculated according to Clinical & Laboratory Standards Institute (CLSI) guideline EP28-A3c. After a baseline exam, a 4-year follow-up survey recorded information about overall morbidity and mortality. The cysC-crea ratio (mean 0.0124 ± 0.0026 mg/μmol) was significantly higher in women and increased progressively with age. Other associated factors were hemoglobin A1c, mean arterial pressure, and C-reactive protein (P < 0.05 for all). Participants exhibiting shrunken pore syndrome had significantly higher ratios of 3.5-66.5 kDa molecules (brain natriuretic peptide, parathyroid hormone, β2-microglobulin, cystatin C, retinol-binding protein, thyroid-stimulating hormone, α1-acid glycoprotein, lipase, amylase, prealbumin, and albumin) and creatinine. There was no such difference in the ratios of very low-molecular weight molecules (urea, uric acid) to creatinine or in the ratios of molecules larger than 66.5 kDa (transferrin, haptoglobin) to creatinine. The cysC-crea ratio was significantly predictive of mortality and subjective overall morbidity at follow-up in logistic regression models adjusting for several factors. The cysC-crea ratio exhibits age- and sex-specific reference intervals in seniors. In conclusion, the cysC-crea ratio may indicate the relative retention of biologically active low-molecular weight compounds and can independently predict the risk for overall mortality and morbidity in the elderly.

Anticoagulation Management Practices and Outcomes in Elderly Patients with Acute Venous Thromboembolism: A Clinical Research Study.

Whether anticoagulation management practices are associated with improved outcomes in elderly patients with acute venous thromboembolism (VTE) is uncertain. Thus, we aimed to examine whether practices recommended by the American College of Chest Physicians guidelines are associated with outcomes in elderly patients with VTE. We studied 991 patients aged ≥65 years with acute VTE in a Swiss prospective multicenter cohort study and assessed the adherence to four management practices: parenteral anticoagulation ≥5 days, INR ≥2.0 for ≥24 hours before stopping parenteral anticoagulation, early start with vitamin K antagonists (VKA) ≤24 hours of VTE diagnosis, and the use of low-molecular-weight heparin (LMWH) or fondaparinux. The outcomes were all-cause mortality, VTE recurrence, and major bleeding at 6 months, and the length of hospital stay (LOS). We used Cox regression and lognormal survival models, adjusting for patient characteristics. Overall, 9% of patients died, 3% had VTE recurrence, and 7% major bleeding. Early start with VKA was associated with a lower risk of major bleeding (adjusted hazard ratio 0.37, 95% CI 0.20-0.71). Early start with VKA (adjusted time ratio [TR] 0.77, 95% CI 0.69-0.86) and use of LMWH/fondaparinux (adjusted TR 0.87, 95% CI 0.78-0.97) were associated with a shorter LOS. An INR ≥2.0 for ≥24 hours before stopping parenteral anticoagulants was associated with a longer LOS (adjusted TR 1.2, 95% CI 1.08-1.33). In elderly patients with VTE, the adherence to recommended anticoagulation management practices showed mixed results. In conclusion, only early start with VKA and use of parenteral LMWH/fondaparinux were associated with better outcomes.

Mutation Update of the CLCN5 Gene Responsible for Dent Disease 1

Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC-5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5-phosphatase OCRL-1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in-frame mutations described were mapped onto a three-dimensional homology model of the ClC-5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies.

The Chemical Mechanism of a Brown-Rot Decay Mimtic System and its Application in Paper Recycling Processes

This work is aimed at improving our current knowledge of the non-enzymatic inecl~anisins involved in brown-rot decay, as well as the exploration of potential applications of a brown-rot mimetic model system in paper recycling processes. The study was divided into two parts. The first part focussed on the chemical mechanisms involved in chelation and reduction of iron by a low molecular weight chelator (isolated from the brown-rot fungus Gloeophyllz~m tmbeum) and its model compound 2,3- dihydroxybenzoic acid (2,3-DHBA). Chelation as well as free radical generation mediated by this system were studied by ESR measurement. The results indicate that the effects of the chelator/iron ratio, the pH, and other reaction parameters on hydroxyl radical generation by a Fenton type system could be determined using ESR spin-trapping techniques. The results also support the hypothesis that superoxide radicals are involved in the chelator-mediated Fenton process. In the second part of the study, the effect of a chelator-mediated Fenton system for the improvement of deinking efficiency and the n~odification of fiber and paper properties was studied. For the deinking study, copy paper was laser printed with an identical standard pattern. Then repulping and flotation operations were performed to remove ink particles. Under properly controlled deinking conditions, the chelator mediated treatment (CMT) resulted in a reduction in dirt count over that of conventional deinking procedures with no significant loss of pulp strength. To study the effect of the chelator system treatment on the quality of pulp with different fines content, a fully bleached hardwood kraft pulp was beaten to different freeness levels and treated with the chelator-mediated free radical system. The result shows that virgin fiber and heavily beaten fiber respond differently to the free radical treatment. Unbeaten fibers become more flexible and easier to collapse after free radical treatment, while beaten fibers show a reduction in fines and small materials after mild free radical treatment.

Study of Basic Wood Decay Mechanisms and Their Biotechnological Applications

The overall objective of this thesis was to gain further understanding of the non-enzymatic mechanisms involved in brown-rot wood decay, especially the role of pH, oxalic acid, and low molecular catecholate compounds on the dissolution and reduction of iron, and the formation of reactive oxygen species. Another focus of this study will be the potential application of a biomimetic free radical generating system inspired from fungi wood decay process, especially the non-enzymatic mechanism. The possible pathways of iron uptake and iron redox cycling in non-enzymatic brown-rot decay were investigated in this study. UV-Vis spectroscopy and HPLC were employed to study the kinetics and pathways of the interaction between iron and model catecholate compounds under different pH and chelator/iron molar ratio conditions. Iron chelation and reduction during early non-enzymatic wood decay processes have been studied in this thesis. The results indicate that the effects of the chelator/iron ratio, the pH, and other reaction parameters on the hydroxyl radical generation in a Fenton type system can be determined using ESR spin-trapping techniques. Data also support the hypothesis that superoxide radicals are involved in chelator-mediated Fenton processes. The mechanisms involved in free radical activation of Thermal Mechanical Pulp fibers were investigated. The activation of TMP fibers was evaluated by ESR measurement of free phenoxy radical generation on solid fibers. The results indicate that low molecular weight chelators can improve Fenton reactions, thus in turn stimulating the free radical activation of TMP fibers. A mediated Fenton system was evaluated for decolorization of several types of dyes. The result shows that the Fenton system mediated by a catecholate-type chelator effectively reduced the color of a diluted solution of synthetic dyes after 90 minutes of treatment at room temperature. The results show that compared to a neat Fenton process, the mediated Fenton decolorization process increased the production, and therefore the effective longevity, of hydroxyl radical species to increase the decolorization efficiency.

METALLOTHIONEIN RECEPTOR EXPRESSION IN LEUKOCYTES

Metallothionein (MT) represents a family of low molecular weight, cysteine-rich proteins that play a number of roles in cellular homeostasis. MT is synthesized as a consequence of a variety of cellular stressors, including exposure to toxic metals, increased temperature, tissue wounding, as well as inflammatory and tumorigenic agents. This protein has been found in both intracellular compartments and extracellular spaces, and its function may depend in part on its location. Extracellular MT is able to redistribute heavy metals between tissues, act as a powerful antioxidant, affect cell proliferation, and cause the suppression of T-dependent humoral immunity. The nature of the interaction of MT with the plasma cell membrane has yet to be characterized, despite many observations that there is a significant pool of extracellular MT, and that this extracellular MT will bind to leukocyte plasma membranes. In light of studies that MT can be detected on the surface of leukocytes from animals immunized in the presence of adjuvant, and that an MT specific receptor has been found on the surface of astrocytes, we have investigated the nature of the potential MT-specific surface receptor-binding site(s) on the plasma membrane of leukocytes. The identification of MT-receptors will allow for the characterization of the mechanism MT uses for immunomodulation, for the manipulation of MT in its immunomodulatory role, and for the identification of patients at higher risk for those potentially harmful immunomodulatory effects.

POU domain transcription factor Brn3b is critical for the normal development and survival of retinal ganglion cells

The POU domain transcription factor Brn3b/POU4F2 plays a critical role regulating gene expression in mouse retinal ganglion cells (RGCs). Previous investigations have shown that Brn3b is not required for initial cell fate specification or migration; however, it is essential for normal RGC differentiation. In contrast to wild type axons, the mutant neurites were phenotypically different: shorter, rougher, disorganized, and poorly fasciculated. Wild type axons stained intensely with axon specific marker tau-1, while mutant projections were weakly stained and the mutant projections showed strong labeling with dendrite specific marker MAP2. Brn-3b mutant axonal projections contained more microtubules and fewer neurofilaments, a dendritic characteristic, than the wild type. The mutant neurites also exhibited significantly weaker staining of neurofilament low-molecular-weight (NF-L) in the axon when compared to the wild type, and NF-L accumulation in the neuron cell body. The absence of Brn-3b results in an inability to form normal axons and enhanced apoptosis in RGCs, suggesting that Brn-3b may control a set of genes involved in axon formation. ^ Brn3b contains several distinct sequence motifs: a glycine/serine rich region, two histidine rich regions, and a fifteen amino acid conserved sequence shared by all Brn3 family members in the N-terminus and a POU specific and POU homeodomain in the C-terminus. Brn3b activates a Luciferase reporter over 25 fold in cell culture when binding to native brn3 binding sites upstream of a minimal promoter. When fused to the Gal4 DNA Binding domain (DBD) and driven by either a strong (CMV) or weaker (pAHD) promoter, the N-terminal of Brn3b is capable of similar activation when binding to Gal4 UAS sites, indicating a presumptive activator of transcription. Both full length Brn3b or the C-terminus fused to the Gal4DBD and driven by pCMV repressed a Luciferase reporter downstream of UAS binding sites. Lower levels of expression of the fusion protein driven by pADH resulted in an alleviation of repression. This repression appears to be a limitation of this system of transcriptional analysis and a potential pitfall in conventional pCMV based transfection assays. ^

Cellular Uptake of Neutrohpil Elastase Links Inflammation to Adaptive Immunity

Many tumors arise from sites of inflammation providing evidence that innate immunity is a critical component in the development and progression of cancer. Neutrophils are primary mediators of the innate immune response. Upon activation, an important function of neutrophils is release of an assortment of proteins from their granules including the serine protease neutrophil elastase (NE). The effect of NE on cancer has been attributed primarily to its ability to degrade the extracellular matrix thereby promoting invasion and metastasis. Recently, it was shown that NE could be taken up by lung cancer cells leading to degradation of insulin receptor substrate-1 thereby promoting hyperactivity of the phosphatidylinositol-3 kinase (PI3K) pathway and tumor cell proliferation. To our knowledge, nobody has investigated uptake of NE by other tumor types. In addition, NE has broad substrate specificity suggesting that uptake of NE by tumor cells could impact processes regulating tumorigenensis other than activation of the PI3K pathway. Neutrophil elastase has been identified in breast cancer specimens where high levels of NE have prognostic significance. These studies have assessed NE levels in whole tumor lysates. Because the major source of NE is from activated neutrophils, we hypothesized that breast cancer cells do not have endogenous NE but may take up NE released by tumor associated neutrophils in the tumor microenvironment and that this could provide a link between the innate immune response to tumors and specific adaptive immune responses. In this thesis, we show that breast cancer cells lack endogenous NE expression and that they are able to take up NE resulting in increased generation of low molecular weight cyclin E (CCNE) and enhanced susceptibility to lysis by CCNE-specific cytotoxic T lymphocytes. We also show that after taking up NE and proteinase 3 (PR3), a second primary granule protease with significant homology to NE, breast cancer cells cross-present the NE- and PR3-derived peptide PR1 rendering them susceptible to PR1-targeted therapies. Taken together, our data support a role for NE uptake in modulating adaptive immune responses against breast cancer.

Proceedings of the 22nd Annual Biochemical Engineering Symposium

This is the twenty-second of a series of symposia devoted to talks and posters by students about their biochemical engineering research. The first, third, fifth, ninth, twelfth, sixteenth, and twenti~th were hosted by Kansas State University, the second and fourth by the University of Nebraska- Lincoln, the sixth, seventh, tenth, thirteenth, seventeenth, and twenty-second by Iowa State University, the eighth, fourteenth, and nineteenth by the University of Missouri-Columbia, the eleventh, fifteenth, and twenty-first by Colorado State University, and the eighteenth by the University of Colorado. Next year's symposium will be at the University of Oklahoma. Symposium proceedings are edited and issued by faculty of the host institution. Because final publication usually takes place in refereed journals, articles included here are brief and often cover work in progress. ContentsC. A. Baldwin, J.P. McDonald, and L. E. Erickson, Kansas State University. Effect of Hydrocarbon Phase on Kinetic and Transport Limitations for Bioremediation of Microporous Soil J. C. Wang, S. K. Banerji, and Rakesh Bajpai, University of Missouri-Columbia. Migration of PCP in Soil-Columns in Presence of a Second Organic Phase Cheng-Hsien Hsu and Roger G. Harrison, University of Oklahoma. Bacterial Leaching of Zinc and Copper from Mining Wastes James A. Searles, Paul Todd, and Dhinakar S. Kompala, University of Colorado. Suspension Culture of Chinese Hamster Ovary Cells Utilizing Inclined Sedimentation Ron Beyerinck and Eric H. Dunlop, Colorado State University. The Effect of Feed Zone Turbulence as Measured by Laser Doppler Velocimetry on Baker's Yeast Metabolism in a Chemostat Paul Li-Hong Yeh, GraceY. Sun, Gary A. Weisman, and Rakesh Bajpai, University of Missouri-Columbia. Effect of Medium Constituents upon Membrane Composition of Insect Cells R. Shane Gold, M. M. Meagher, R. Hutkins, and T. Conway, University of Nebraska-Lincoin. Ethanol Tolerance and Carbohydrate Metabolism in Lactobacilli John Sargantanis and M. N. Karim, Colorado State University. Application of Kalman Filter and Adaptive Control in Solid Substrate Fermentation D. Vrana, M. Meagher, and R. Hutkins, University of Nebraska-Lincoln. Product Recovery Optimization in the ABE Fermentation Kalyan R. Tadikonda and Robert H. Davis, University of Colorado. Cell Separations Using Targeted Monoclonal Antibodies Against Surface Proteins Meng H. Heng and Charles E. Glatz, Iowa State University. Charged Fusion for Selective Recovery of B-Galactosidase from Cell Extract Using Hollow Fiber Ion-Exchange Membrane Adsorption Hsiu-Mei Chen, Peter J. Reilly, and Clark Ford, Iowa State University. Site-Directed Mutagenesis to Enhance Thermostability of Glucoamylase from Aspergillus: A Rational Approach P. Tuitemwong, L. E. Erickson, and D. Y. C. Fung, Kansas State University. Applications of Enzymatic Hydrolysis and Fermentation on the Reduction of Flatulent Sugars in the Rapid Hydration Hydrothermal Cooked Soy Milk Sanjeev Redkar and Robert H. Davis, University of Colorado. Crossflow Microfiltration of Yeast Suspensions Linda Henk and James C. Linden, Colorado State University, and Irving C. Anderson, Iowa State University. Evaluation of Sorghum Ensilage as an Ethanol Feedstock Marc Lipovitch and James C. Linden, Colorado State University. Stability and Biomass Feedstock Pretreatability for Simultaneous Saccharification and Fermentation Ali Demirci, Anthony L. Pometto Ill, and Kenneth E. Johnson, Iowa State University. Application of Biofilm Reactors in Lactic Acid Fermentation Michael K. Dowd, Peter I. Reilly, and WalterS. Trahanovsky, Iowa State University. Low Molecular-Weight Organic Composition of Ethanol Stillage from Corn Craig E. Forney, Meng H. Heng, John R. Luther, Mark Q. Niederauer, and Charles E. Glatz, Iowa State University. Enhancement of Protein Separation Using Genetic Engineering J. F. Shimp, J. C. Tracy, E. Lee, L. C. Davis, and L. E. Erickson, Kansas State University. Modeling Contaminant Transport, Biodegradation and Uptake by Plants in the Rhizosphere Xiaoqing Yang, L. E. Erickson, and L. T. Fan, Kansas State University. Modeling of Dispersive-Convective Characteristics in Bioremediation of Contaminated Soil Jan Johansson and Rakesh Bajpai, University of Missouri-Columbia. Fouling of Membranes J. M. Wang, S. K. Banerji, and R. K. Bajpai, University of Missouri-Columbia. Migration of Sodium-Pentachorophenol (Na-PCP) in Unsaturated and Saturated Soil-Columns J. Sweeney and M. Meagher, University of Nebraska-Lincoln. The Purification of Alpha-D-Glucuronidase from Trichoderma reesei

Proceedings of the 24th Annual Biochemical Engineering Symposium

The 24th Biochemical Engineering Symposium was held 9-10 September 1994 at the YMCA of the Rockies conference center in Estes Park, Colorado, under the sponsorship of the Department of Chemical Engineering at the University of Colorado. Previous symposia in this series have been hosted by Kansas State University (1st, 3rd, 5th, 9th, 12th, 16th, 20th), University of Nebraska-Lincoln (2nd, 4th), Iowa State University (6th, 7th, 10th, 13th, 17th, 22nd), University of Missouri-Columbia (8th, 14th, 19th), Colorado State University (11th, 15th, 21st), University of Colorado (18th), and the University of Oklahoma (23rd). The next symposium is scheduled to be held at the University of Missouri-Columbia. The symposia are devoted to talks by students about their ongoing research. Because final publication usually takes place elsewhere, the papers included in the proceedings are brief, and often cover work in progress. ContentsIn-Well Aeration: An Innovative Subsurface Remediation TechnologyPrashant Gandhi, X. Yang, L.E. Erickson, and L. T. Fan; Kansas State University Expression of an Antimicrobial Peptide Analog in Eacherlchill coliChris Haught and Roger G. Harrison; University of Oklahoma Using High-frequency Backpulaing to Maximize Croasflow Filtration PerformanceSanjeev G. Redkar and Robert H. Davis; University of Colorado Low Molecular Weight Organic Compositions of Acid Waters from Vegetable Oil SoapstocksSteven L. Johansen, Arunthathi Sivasothy, Peter J. Reilly, and Earl G. Hammond; Iowa State University; Michael K. Dowd; U.S. Department of Agriculture Gas Phase Composition Effects on Suspension Cultures of Taxus cuspidata Noushin Mirjalili and James C. Linden; Colorado State University Cybernetic Modeling of Spontaneous Oscillations in Continuous Cultures of Ssccharomyces cerevisiaeKenneth D. Jones and Dhinakar S. Kompala; University of Colorado The Effect of Turbulent Shear on Calcium Mobilization in Mammalian CellsChristopher M. Cannizzaro, Pradyumna K. Namdev, and Eric H. Dunlop; Colorado State University Experimental Studies of Droplet Ejection at the Free Surface In Sparged ReactorsT. Y. Yiin, L A. Glasgow, and L. E. Erickson; Kansas State University The Role of Domain E (Starch-Binding Region) on the Activity of a Bacillus macersns Cyclodextrln GlucanotransferaseHai-yin Chang, Trang Le, and Zivko L. Nikolov; Iowa State University Use of the Rotating Wall Vessel for Study of Plant Cell Suspension CulturesXinzhi Sun and James C. Linden; Colorado State University A Novel Counter-Current Distribution Apparatus for the Study of Multi-Stage Aqueous Two-Phase Extraction of Biomolecules and Cell ParticlesMartin R. Guinn and Paul Todd; University of Colorado The Dynamics of Unhooking and Contraction of a Polyelectrolyte Chain Around an Isolated PostLin Zhang and Edith M. Sevick; University of Colorado A Laboratory Study of the Fate of Trichloroathylene and 1,1,1-Trlchloroathane In the Presence of Alfalfa PlantsMuralidharan Narayanan, Ryan M. Green, Lawrence C. Davis, and Larry E. Erickson; Kansas State University Modeling the Fate of Pyrene In the RhIzosphereS.K. Santharam, LE. Erickson, and L. T. Fan; Kansas State University Derivatization of MaltooligosaccharidesDaniela Prinz, Peter J. Reilly, and Zivko L. Nikolov; Iowa State University Probing Surfactant-Protein Binding by EPA SpectroscopyNarendra B. Bam, Yale University; Theodore W. Randolph; University of Colorado Optimization of a Stir-Cell Bioreactor for In Vitro Production of RNANeal T. Williams, Kim A. Wicklund, and Robert H. Davis; University of Colorado

Concentrations and carbon isotopic compositions of organic and inorganic compounds in sediment porewaters at IODP Site U1329

Ocean drilling has revealed the existence of vast microbial populations in the deep subseafloor, but to date little is known about their metabolic activities. To better understand the biogeochemical processes in the deep biosphere, we investigate the stable carbon isotope chemistry of acetate and other carbon-bearing metabolites in sediment pore-waters. Acetate is a key metabolite in the cycling of carbon in anoxic sediments. Its stable carbon isotopic composition provides information on the metabolic processes dominating acetate turnover in situ. This study reports our findings for a methane-rich site at the northern Cascadia Margin (NE Pacific) where Expedition 311 of the Integrated Ocean Drilling Program (IODP) sampled the upper 190 m of sediment. At Site U1329, d13C values of acetate span a wide range from -46.0 per mill to -11.0 per mill vs. VPDB and change systematically with sediment depth. In contrast, d13C values of both the bulk dissolved organic carbon (DOC) (-21.6 ± 1.3 per mill vs. VPDB) and the low-molecular-weight compound lactate (-20.9 ± 1.8 per mill vs. VPDB) show little variability. These species are interpreted to represent the carbon isotopic composition of fermentation products. Relative to DOC, acetate is up to 23.1 per mill depleted and up to 9.1 per mill enriched in 13C. Broadly, 13C-depletions of acetate relative to DOC indicate flux of carbon from acetogenesis into the acetate pool while 13C-enrichments of pore-water acetate relative to DOC suggest consumption of acetate by acetoclastic methanogenesis. Isotopic relationships between acetate and lactate or DOC provide new information on the carbon flow and the presence and activity of specific functional microbial communities in distinct biogeochemical horizons of the sediment. In particular, they suggest that acetogenic CO2-reduction can coexist with methanogenic CO2-reduction, a notion contrary to the hypothesis that hydrogen levels are controlled by the thermodynamically most favorable electron-accepting process. Further, the isotopic relationship suggests a relative increase in acetate flow to acetoclastic methanogenesis with depth although its contribution to total methanogenesis is probably small. Our study demonstrates how the stable carbon isotope biogeochemistry of acetate can be used to identify pathways of microbial carbon turnover in subsurface environments. Our observations also raise new questions regarding the factors controlling acetate turnover in marine sediments.

Abundances and d13C values of n-alkanes extracted from sediment samples of ODP Hole 116-717C

Siwalik paleosol and Bengal Fan sediment samples were analyzed for the abundance and isotopic composition of n-alkanes in order to test for molecular evidence of the expansion of C4 grasslands on the Indian subcontinent. The carbon isotopic compositions of high-molecular-weight alkanes in both the ancient soils and sediments record a shift from low d13C values (ca. -30 per mil) to higher values (ca. -22 per mil) prior to 6 Ma. This shift is similar in magnitude to that recorded by paleosol carbonate and fossil teeth, and is consistent with a relatively rapid transition from dominantly C3 vegetation to an ecosystem dominated by C4 plants typical of semi-arid grasslands. The n-alkane values from our paleosol samples indicate that the isotopic change began as early as 9 Ma, reflecting either a growing contribution of C4 plants to a dominantly C3 biomass or a decrease in water availability to C3 plants. Molecular and isotopic analyses of other compounds, including n-alcohols and low-molecular weight n-alkanes indicate paleosol organic matter contains contributions from a mixture of sources, including vascular plants, algae and/or cyanobacteria and microorganisms. A range of inputs is likewise reflected in the isotopic composition of the total organic carbon from these samples. In addition, the n-alkanes from two samples show little evidence for pedegenic inputs and we suggest the compounds were derived instead from the paleosol's parent materials. We suggest the record of vegetation in ancient terrestrial ecosystems is better reconstructed using isotopic signatures of molecular markers, rather than bulk organic carbon. This approach provides a means of expanding the spatial and temporal records of C4 plant biomass which will help to resolve possible tectonic, climatic or biological controls on the rise of this important component of the terrestrial biosphere.

Methanol and ethanol analysis in marine sediment pore water

Low-molecular-weight (LMW) alcohols are produced during the microbial degradation of organic matter from precursors such as lignin, pectin, and carbohydrates. The biogeochemical behavior of these alcohols in marine sediment is poorly constrained but potentially central to carbon cycling. Little is known about LMW alcohols in sediment pore waters because of their low concentrations and high water miscibility, both of which pose substantial analytical challenges. In this study, three alternative methods were adapted for the analysis of trace amounts of methanol and ethanol in small volumes of saline pore waters: direct aqueous injection (DAI), solid-phase microextraction (SPME), and purge and trap (P&T) in combination with gas chromatography (GC) coupled to either a flame ionization detector (FID) or a mass spectrometer (MS). Key modifications included the desalination of samples prior to DAI, the use of a threaded midget bubbler to purge small-volume samples under heated conditions and the addition of salt during P&T. All three methods were validated for LMW alcohol analysis, and the lowest detection limit (60 nM and 40 nM for methanol and ethanol, respectively) was achieved with the P&T technique. With these methods, ambient concentrations of volatile alcohols were determined for the first time in marine sediment pore waters of the Black Sea and the Gulf of Mexico. A strong correlation between the two compounds was observed and tentatively interpreted as being controlled by similar sources and sinks at the examined stations.