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Introduced seed dispersers can shift seed predation dynamics in native species, impacting native plant establishment and spread. We studied the effect of the non-native earthworm, Lumbricus terrestris, on seed loss dynamics in the large-seeded native annual, Ambrosia trifida (giant ragweed), an expanding agricultural weed whose seeds are heavily predated by mice. Lumbricus terrestris may protect seeds against rodent predation by caching them in its burrows from which seedlings can emerge unharmed; however, little is known about the ability of earthworms to compete with rodents for seeds. We investigated this interaction and how environmental factors affected the competitive outcome. In a 2-year field study, we analysed relative rates of seed removal by earthworms and mice for seeds dispersed at various times in habitats that varied in vegetative cover. Species-specific responses to environmental conditions drove variation in the share of seeds taken by earthworms versus mice, with earthworms gaining relatively more seeds under warmer, wetter conditions and in low plant cover habitats, and mice obtaining more seeds under colder, drier conditions and in high cover habitats. Environmental factors also determined which competitor accessed seeds first, and this conferred a competitive advantage that was compounded over time. Earthworms cached some seeds under all experimental conditions, suggesting that L. terrestris can act mutualistically with giant ragweed in diverse environments. Synthesis and applications. Our results support the hypothesis that the non-native earthworm Lumbricus terrestris behaves as a seed dispersal mutualist for the native annual giant ragweed by caching its seeds in its burrows, thereby reducing their availability to rodent seed predators. The data also support the view that interactions among the environment and competing seed predators determine the fate of seed pools in response to species-specific environmental preferences. In crop fields where L. terrestris is abundant, additional efforts to prevent giant ragweed seed return may be warranted due to the likelihood that earthworms will increase giant ragweed soil seed pools. Lumbricus terrestris is expanding its range throughout temperate regions and may similarly affect secondary seed dispersal and seed predation dynamics of large-seeded species in other plant communities. National Research Initiative of the USDA Cooperative State Research, Education and Extension ServiceUnited States Department of Agriculture (USDA)National Institute of Food and Agriculture [2005-02261]; USDA National Institute of Food and AgricultureUnited States Department of Agriculture (USDA) [102183] Published version The authors thank Renata Harrison for seed tagging methodology; Brian Schutte, Ramarao Venkatesh and Norman Arancon for fieldwork assistance; and Kristin Mercer and two anonymous reviewers for reviewing early drafts. This manuscript is dedicated in the memory of our recently deceased colleague and mentor in earthworm biology, Clive A. Edwards. This project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant no. 2005-02261 and by the USDA National Institute of Food and Agriculture, grant no. Hatch Project 102183.

Spatial patterns of trait variation across a species' range have implications for population success and evolutionary change potential, particularly in range-expanding and weedy species that encounter distinct selective pressures at large and small spatial scales simultaneously. We investigated intraspecific trait variation in a common garden experiment with giant ragweed (Ambrosia trifida), a highly variable agricultural weed with an expanding geographic range and broad ecological amplitude. Our study included paired populations from agricultural and natural riparian habitats in each of seven regions ranging east to west from the core of the species' distribution in central Ohio to southeastern Minnesota, which is nearer the current invasion front. We observed trait variation across both large- and small-scale putative selective gradients. At large scales, giant ragweed populations from the westernmost locations were nearly four times more fecund and had a nearly 50% increase in reproductive allocation compared to populations from the core. The degree of surface texture on fruits also declined from east to west. Greater fecundity in the west represents a putative trade-off between fruit size and fruit number across the study region, although no such trade-off was found across individual plants. This pattern may effectively result in greater propagule pressure closer to the invasion front. At smaller spatial scales, plants from agricultural populations emerged later and were smaller than plants from riparian populations. However, because plants from agricultural populations allocated more biomass to reproduction, total fecundity did not differ across habitats. Our emergence data are consistent with previous observations showing delayed emergence in agricultural compared to natural populations; thus evolutionary change may be predictable as giant ragweed continues spreading into agricultural fields throughout North America. These shifts in life-history strategy apparently bear no fecundity cost, suggesting that giant ragweed's success can be attributed at least in part to its substantial adaptive potential.

The objective in routine analyses is generally to determine a small number of analytes. With samples containing ~103 or more components there will be insufficient peak capacity to resolve analytes from nonanalytes. This issue was addressed herein through a new type of separation mechanism in which small groups of targeted analytes are bound with high affinity to a soluble analyte-sequestering transport phase (ASTP) composed of a ~25 nm Stokes radius hydrophilic polymer core (HPC). When introduced into a 30 nm pore diameter size-exclusion chromatography (SEC) column, ASTP/analyte complexes elute within minutes, together, unretained, and relatively pure in the first chromatographic peak. Nonanalytes, in contrast, enter pore matrices of the packing material, are retarded in elution velocity, and are eluted later, separated from analytes. Fabrication of ASTPs was achieved by covalently coupling an antibody or some other affinity selector to a high molecular weight HPC. Beyond sequestering analytes, the function of ASTPs is to act as a molecular weight shifting agent, conveying an effective molecular weight to analytes that is much larger than that of nonanalytes and causing them to elute in the SEC void volume. This mode of separation is referred to as mobile affinity sorbent chromatography (MASC). Subsequent to their purification, ASTP/analyte complexes were detected by fluorescence spectrometry.

Field studies were conducted in 2011 and 2012 to compare mulch treatments of shredded newspaper, a combination of shredded newspaper plus turfgrass clippings (NP + grass), hardwood bark chips, black polyethylene plastic, and bare soil on weeds, insects, soil moisture, and soil temperature in pumpkins. Newspaper mulch or black plastic reduced total weed biomass ≥90%, and woodchip or NP + grass mulch each reduced total weed biomass 78% compared with bare soil under high rainfall conditions in 2011. In 2012, under low rainfall, all mulches reduced weed biomass 97% or more compared with bare soil. In both years, all mulches resulted in higher squash bug infestations than bare soil. The woodchip, newspaper, and NP + grass mulches retained higher soil moistures than bare soil or black plastic over the course of each growing season, and the woodchip and NP + grass mulches caused greatest fluctuations in soil temperature. Pumpkin yields were abnormally low in 2011 and did not differ among treatments. In 2012, all mulches produced greater total marketable pumpkin fruit weights compared with bare soil, but only black plastic, newspaper, and NP + grass mulches resulted in greater total numbers of marketable pumpkins. Overall results indicate that shredded newspaper or NP + grass mulches may be useful for organic and/or small-scale urban crop producers as sustainable alternatives to black plastic mulch; however, their weed suppression efficacy may require higher application rates with increasing moisture conditions, and they may require greater squash bug control measures than under bare soil conditions.

Giant ragweed has been increasing as a major weed of row crops in the last 30 yr, but quantitative data regarding its pattern and mechanisms of spread in crop fields are lacking. To address this gap, we conducted a Web-based survey of certified crop advisors in the U.S. Corn Belt and Ontario, Canada. Participants were asked questions regarding giant ragweed and crop production practices for the county of their choice. Responses were mapped and correlation analyses were conducted among the responses to determine factors associated with giant ragweed populations. Respondents rated giant ragweed as the most or one of the most difficult weeds to manage in 45% of 421 U.S. counties responding, and 57% of responding counties reported giant ragweed populations with herbicide resistance to acetolactate synthase inhibitors, glyphosate, or both herbicides. Results suggest that giant ragweed is increasing in crop fields outward from the east-central U.S. Corn Belt in most directions. Crop production practices associated with giant ragweed populations included minimum tillage, continuous soybean, and multiple-application herbicide programs; ecological factors included giant ragweed presence in noncrop edge habitats, early and prolonged emergence, and presence of the seed-burying common earthworm in crop fields. Managing giant ragweed in noncrop areas could reduce giant ragweed migration from noncrop habitats into crop fields and slow its spread. Where giant ragweed is already established in crop fields, including a more diverse combination of crop species, tillage practices, and herbicide sites of action will be critical to reduce populations, disrupt emergence patterns, and select against herbicide-resistant giant ragweed genotypes. Incorporation of a cereal grain into the crop rotation may help suppress early giant ragweed emergence and provide chemical or mechanical control options for late-emerging giant ragweed.

Background:Regulatory T cells (Tregs) prevent autoimmunity and control inflammation. As low-dose interleukin-2 (ld-IL2) expands and activates Tregs, it has a broad therapeutic potential for any autoimmune or inflammatory disease (AIID). We performed a disease-finding “basket trial” (TRANSREGNCT01988506) in patients affected by one of 11 different AIID and reported the outcome of the first 46 patients (Rosenzwajg et al, ARD 2019).Objectives:Here we analyzed and discussed results from deep immunophenotyping, of 78 patients, to comprehensively study the effect of ld-IL2 on the immune system of patients affected by various AIIDMethods:We performed a prospective, open label, phase I-IIa study in 78 patients with a mild to moderate form of one of 13 selected AIID. All patients received ld-IL2 (1 million IU/day) for 5 days, followed by fortnightly injections for 6 months. Deep immunophenotyping was performed before and after 5 days of ld-IL2.Results:ld-IL2 significantly expands both memory Tregs as well as naïve Tregs, including recent thymic emigrant Tregs. It also activates Tregs as demonstrated by the significantly increased expression of HLA-DR, CD39, CD73, GITR, CTLA-4. Similar results were observed across the different AIID.Conclusion:ld-IL2 “universally” improves Treg fitness across 13 autoimmune and inflammatory disease.References:[1]Rosenzwajg M#, Lorenzon R#, Cacoub P, Pham HP, Pitoiset F, El Soufi K, RIbet C, Bernard C, Aractingi S, Banneville B, Beaugerie L, Berenbaum F, Champey J, Chazouilleres O, Corpechot C, Fautrel B, Mekinian A, Regnier E, Saadoun D, Salem JE, Sellam J, Seksik P, Daguenel-Nguyen A, Doppler V, Mariau J, Vicaut E, Klatzmann D. Immunological and clinical effects of low-dose interleukin-2 across 11 autoimmune diseases in a single, open clinical trial. Ann Rheum Dis. 2019 Feb;78(2):209-217. doi: 10.1136/annrheumdis-2018-214229. Epub 2018 Nov 24.Disclosure of Interests:Michelle Rosenzwajg: None declared, Roberta Lorenzon: None declared, Patrice cacoub: None declared, Fabien Pitoiset: None declared, Selim Aractingi: None declared, Beatrice Banneville Speakers bureau: Lilly, Novartis, Laurent Beaugerie: None declared, Francis Berenbaum Grant/research support from: TRB Chemedica (through institution), MSD (through institution), Pfizer (through institution), Consultant of: Novartis, MSD, Pfizer, Lilly, UCB, Abbvie, Roche, Servier, Sanofi-Aventis, Flexion Therapeutics, Expanscience, GSK, Biogen, Nordic, Sandoz, Regeneron, Gilead, Bone Therapeutics, Regulaxis, Peptinov, 4P Pharma, Paid instructor for: Sandoz, Speakers bureau: Novartis, MSD, Pfizer, Lilly, UCB, Abbvie, Roche, Servier, Sanofi-Aventis, Flexion Therapeutics, Expanscience, GSK, Biogen, Nordic, Sandoz, Regeneron, Gilead, Sandoz, Julien Champey: None declared, Olivier Chazouilleres: None declared, Christophe Corpechot: None declared, Bruno Fautrel Grant/research support from: AbbVie, Lilly, MSD, Pfizer, Consultant of: AbbVie, Biogen, BMS, Boehringer Ingelheim, Celgene, Lilly, Janssen, Medac MSD France, Nordic Pharma, Novartis, Pfizer, Roche, Sanofi Aventis, SOBI and UCB, Arsene Mekinian: None declared, Elodie Regnier: None declared, david Saadoun: None declared, Joe-Elie Salem: None declared, Jérémie SELLAM: None declared, Philippe Seksik: None declared, David Klatzmann Consultant of: ILTOO Pharma

Zucchini squash (Cucurbita pepo) has a high pollination demand, and the native, ground-nesting squash bee (Peponapis pruinosa) provides the majority of the crop’s pollination requirement in some environments. Squash bees nest directly in crop fields, and nests can be disturbed by tillage and other management operations. Mulches that use municipal waste materials may provide a weed control strategy for squash plantings that is more benign to squash bees than cultivation. Field and greenhouse studies were conducted in 2011 and 2012 to compare the effects of nontillage weed control methods including polyethylene black plastic, woodchips, shredded newspaper, a combination of shredded newspaper plus grass clippings (NP + grass), and bare soil (control) on soil characteristics, squash pollination and fruit production, and squash bee nesting. Woodchips, shredded newspaper, and NP + grass mulch decreased soil temperature, while soils beneath newspaper mulch retained more moisture. Unmarketable, misshapen fruit occurred more frequently in plastic than in the other mulch treatments. No measurable differences in floral resource production or crop pollination were found among treatments, suggesting that misshapen fruit resulted from high soil temperatures in black plastic plots rather than poor pollinator attraction. Squash bee nests were located within bare soil, newspaper, and NP + grass plots, indicating that these mulches did not prevent nesting. NP + grass mulch had a positive effect on plant growth and fruit production, possibly from an addition of plant-available nitrogen or the presence of preferable nesting ground. Shredded newspaper when combined with grass clippings performed as an effective mulch material that improved crop performance with no apparent negative impacts on squash bee nesting or on squash floral resources and pollination.

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n = 5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ~90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNAseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were upregulated in triple negative vs. luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.

Giant ragweed germination is delayed by both a physiological dormancy of the embryo (embryo dormancy) and an inhibitory influence of embryo-covering structures (covering structure-enforced [CSE] dormancy). To clarify the roles of embryo and CSE dormancy in giant ragweed seedling emergence timing, we conducted two experiments to address the following objectives: (1) determine changes in germinability for giant ragweed dispersal units (hereafter “involucres”) and their components under natural burial conditions, and (2) compare embryo and CSE dormancy alleviation and emergence periodicity between successional and agricultural populations. In Experiment 1, involucres were buried in crop fields at Columbus, OH, periodically excavated, and brought to the laboratory for dissection. Involucres, achenes, and embryos were then subjected to germination assays at 20 C. In Experiment 2, temporal patterns of seedling emergence were determined at a common burial site. Reductions in embryo and CSE dormancy were compared with controlled-environment stratification followed by germination assays at 12 and 20 C, temperatures representative of soil conditions in spring and summer. Results indicated that overwinter dormancy loss involved sequential reductions in embryo and CSE dormancy. CSE dormancy, which may limit potential for fatal germination during fall, was caused by the pericarp and/or embryo-covering structures within the pericarp. In Experiment 2, successional populations emerged synchronously in early spring, whereas agricultural populations emerged throughout the growing season. Levels of embryo dormancy were greater in the agricultural populations than the successional populations, but CSE dormancy levels were similar among populations. In 12 C germination assays, embryo dormancy levels were positively correlated with time required to reach 95% cumulative emergence (run 1:r= 0.81, P = 0.03; run 2:r= 0.76, P = 0.05). These results suggest that late-season emergence in giant ragweed involves high levels of embryo dormancy that prevent germination at low temperatures in spring.

The focus of this study was on the assessment of technology that might be of clinical utility in identification, quantification, characterization of carbonylation in human plasma proteins. Carbonylation is widely associated with oxidative stress diseases. Breast cancer patient samples were chosen as a stress positive case based on the fact that oxidative stress has been reported to be elevated in this disease. Measurements of 8-isoprostane in plasma confirmed that breast cancer patients in this study were indeed experiencing significant oxidative stress. Carbonyl groups in proteins from freshly drawn blood were derivatized with biotin hydrazide after which the samples were dialyzed and the biotinylated proteins subsequently selected, digested and labeled with iTRAQ™ heavy isotope coding reagent(s). Four hundred sixty proteins were identified and quantified, 95 of which changed 1.5 fold or more in concentration. Beyond confirming the utility of the analytical method, association of protein carbonylation was examined as well. Nearly one fourth of the selected proteins were of cytoplasmic, nuclear, or membrane origin. Analysis of the data by unbiased knowledge assembly methods indicated the most likely disease associated with the proteins was breast neoplasm. Pathway analysis showed the proteins which changed in carbonylation were strongly associated with Brca1, the breast cancer type-1 susceptibility protein. Pathway analysis indicated the major molecular functions of these proteins are defense, immunity and nucleic acid binding.

Cyclin dependent kinase-5 (Cdk5) activity is deregulated in Alzheimer's disease (AD) and contributes to all three hallmarks: neurotoxic β-amyloid formation, neurofibrillary tangles, and neuronal death. However, the mechanism leading to Cdk5 deregulation remains controversial. Cdk5 deregulation in AD is usually linked to the formation of p25, a proteolysis product of Cdk5 activator p35, which leads to Cdk5 mislocalization and hyperactivation. A few studies have indeed shown increased p25 levels in AD brains; however, others have refuted this observation. These contradictory findings suggest that additional factors contribute to Cdk5 deregulation. This study identified glutathione-S-transferase pi 1 (GSTP1) as a novel Cdk5 regulatory protein. We demonstrate that it is a critical determinant of Cdk5 activity in human AD brains and various cancer and neuronal cells. Increased GSTP1 levels were consistently associated with reduced Cdk5 activity. GSTP1 directly inhibits Cdk5 by dislodging p25/p35, and indirectly by eliminating oxidative stress. Cdk5 promotes and is activated by oxidative stress, thereby engaging a feedback loop which ultimately leads to cell death. Not surprisingly, GSTP1 transduction conferred a high degree of neuroprotection under neurotoxic conditions. Given the critical role of oxidative stress in AD pathogenesis, an increase in GSTP1 level may be an alternative way to modulate Cdk5 signaling, eliminate oxidative stress, and prevent neurodegeneration.

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.

Recent studies have shown that antibodies targeting Lewis x (Le(x)) antigen are a valuable tool in the isolation and identification of glycoproteins in plasma. A focus of this study was to determine whether sialylated Lewis x (sLe(x)) antigen carrying glycoproteins occur in human plasma and whether an antibody targeting this antigen could be used to isolate and identify glycoproteins bearing this antigen. An additional objective was to determine the degree to which proteins conjugated to Le(x) and sLe(x) antigens are similar in structure. A specific anti-sLe(x) antibody (anti-sLe(x)Ab), CHO-131, immobilized in an immunoaffinity column was used to select a set of specific sLe(x) bearing proteins from human plasma, after which they were identified by either of two analytical strategies. One approach was to further resolve the affinity selected proteins by reversed phase chromatography (RPC), tryptic digest the RPC fractions, and identify peptide fragments by MALDI-MS/MS. The second was to tryptic digest the affinity selected protein fraction, further resolve the tryptic fragments by RPC, and identify peptides from RPC fractions by MALDI-MS/MS. Histidine-rich glycoprotein, plasminogen, apolipoprotein A-I, vitronectin, proteoglycan-4, clusterin, Ig gamma-2 chain C region, Ig mu chain C region, and interalpha-trypsin inhibitor heavy chain H4 were found to change three folds or more in association with breast cancer. Fifty percent of the glycoproteins carrying either sLe(x) antigen from CHO-131 selection, Le(x) antigen from selection with TG-1 antibody, or both were found to be changed three folds or more in concentration in breast cancer plasma relative to controls.

Excessive oxidative stress leaves a protein carbonylation fingerprint in biological systems. Carbonylation is an irreversible post-translational modification (PTM) that often leads to the loss of protein function and can be a component of multiple diseases. Protein carbonyl groups can be generated directly (by amino acids oxidation and the alpha-amidation pathway) or indirectly by forming adducts with lipid peroxidation products or glycation and advanced glycation end-products. Studies of oxidative stress are complicated by the low concentration of oxidation products and a wide array of routes by which proteins are carbonylated. The development of new selection and enrichment techniques coupled with advances in mass spectrometry are allowing the identification of hundreds of new carbonylated protein products from a broad range of proteins located at many sites in biological systems. The focus of this review is on the use of proteomics tools and methods to identify oxidized proteins along with specific sites of oxidative damage and the consequences of protein oxidation.

Proteomics is the large-scale study of proteins, particularly their expression, structures and functions. This still-emerging combination of technologies aims to describe and characterize all expressed proteins in a biological system. Because of upper limits on mass detection of mass spectrometers, proteins are usually digested into peptides and the peptides are then separated, identified and quantified from this complex enzymatic digest. The problem in digesting proteins first and then analyzing the peptide cleavage fragments by mass spectrometry is that huge numbers of peptides are generated that overwhelm direct mass spectral analyses. The objective in the liquid chromatography approach to proteomics is to fractionate peptide mixtures to enable and maximize identification and quantification of the component peptides by mass spectrometry. This review will focus on existing multidimensional liquid chromatographic (MDLC) platforms developed for proteomics and their application in combination with other techniques such as stable isotope labeling. We also provide some perspectives on likely future developments.

Background: Cancer has profound effects on gene expression, including a cell’s glycosylation machinery. Thus, tumors produce glycoproteins that carry oligosaccharides with structures that are markedly different from the same protein produced by a normal cell. A single protein can have many glycosylation sites that greatly amplify the signals they generate compared with their protein backbones.Content: In this article, we survey clinical tests that target carbohydrate modifications for diagnosing and treating cancer. We present the biological relevance of glycosylation to disease progression by highlighting the role these structures play in adhesion, signaling, and metastasis and then address current methodological approaches to biomarker discovery that capitalize on selectively capturing tumor-associated glycoforms to enrich and identify disease-related candidate analytes. Finally, we discuss emerging technologies—multiple reaction monitoring and lectin-antibody arrays—as potential tools for biomarker validation studies in pursuit of clinically useful tests.Summary: The future of carbohydrate-based biomarker studies has arrived. At all stages, from discovery through verification and deployment into clinics, glycosylation should be considered a primary readout or a way of increasing the sensitivity and specificity of protein-based analyses.

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35–60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low mug/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.

Summary 1 Ambrosia trifida L. (giant ragweed), an aggressive weed of US grain crops and indigenous to North America, colonizes no-tillage crop fields and undisturbed soils despite its large seed size and susceptibility to seed predation. Secondary seed dispersal is critical to seed survival and seedling establishment, yet mechanisms of secondary dispersal are poorly understood for such large-seeded weedy species. 2 Field experiments were conducted to determine how seed foraging by the European exotic burrowing earthworm Lumbricus terrestris L., affected A. trifida seed burial and seedling recruitment, and to determine seed selectivity by L. terrestris. 3 Earthworms collected and buried over 90% of A. trifida seeds placed on the soil surface at a rate eightfold faster than abiotic seed burial. There was a sixfold higher concentration of seeds in burrows than in surrounding soil and a mean of 127 A. trifida seeds per burrow after a single season of A. trifida seed dispersal. 4 Earthworms buried A. trifida seeds from 0·5 to 22 cm deep and reduced recruitment by 37% compared to seeds buried abiotically and protected from predators, due to burial of some seeds below emergence depth limits. However, seedling biomass was increased by 30%. 5 Earthworms foraged selectively among seeds of 11 large-seeded species and collected more seeds of A. trifida than of other species. The earthworms buried small (8·5 mm) A. trifida seeds more deeply and reduced their emergence more than large (11·5 mm) A. trifida seeds. 6 Synthesis and applications. The novel interaction of L. terrestris and A. trifida increases seed bank formation of A. trifida. Burrows of L. terrestris provide safe sites for seeds of A. trifida that may increase seedling establishment in environments with a high risk of seed predation. Control measures to prevent reproduction by A. trifida should be increased where L. terrestris is present due to the earthworms’ ability to bury and protect weed seeds. Selective seed caching by this widely distributed earthworm species may change plant community composition in agricultural and natural areas in North America and influence the evolution of seed traits. Land managers should consider the effects of L. terrestris on seedling regeneration of native and exotic plant species in areas undergoing colonization by L. terrestris.

It is proposed that conformational changes induced in proteins by oxidation can lead to loss of activity or protein aggregation through exposure of hydrophobic residues and alteration in surface hydrophobicity. Because increased oxidative stress and protein aggregation are consistently observed in amyotrophic lateral sclerosis (ALS), we used a 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (BisANS) photolabeling approach to monitor changes in protein unfolding in vivo in skeletal muscle proteins in ALS mice. We find two major proteins, creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), conformationally affected in the ALS G93A mouse model concordant with a 43% and 41% reduction in enzyme activity, respectively. This correlated with changes in conformation and activity that were detected in CK and GAPDH with in vitro oxidation. Interestingly, we found that GAPDH, but not CK, is conformationally and functionally affected in a longer-lived ALS model (H46R/H48Q), exhibiting a 22% reduction in enzyme activity. We proposed a reaction mechanism for BisANS with nucleophilic amino acids such as lysine, serine, threonine, and tyrosine, and BisANS was found to be primarily incorporated to lysine residues in GAPDH. We identified the specific BisANS incorporation sites on GAPDH in nontransgenic (NTg), G93A, and H46R/H48Q mice using liquid chromatography–tandem mass spectrometry analysis. Four BisANS-containing sites (K52, K104, K212, and K248) were found in NTg GAPDH, while three out of four of these sites were lost in either G93A or H46R/H48Q GAPDH. Conversely, eight new sites (K2, K63, K69, K114, K183, K251, S330, and K331) were found on GAPDH for G93A, including one common site (K114) for H46R/H48Q, which is not found on GAPDH from NTg mice. These data show that GAPDH is differentially affected structurally and functionally in vivo in accordance with the degree of oxidative stress associated with these two models of ALS.

Late-season giant ragweed emergence in Ohio crop fields complicates decisions concerning the optimum time to implement control measures. Our objectives were to develop a hydrothermal time emergence model for a late-emerging biotype and validate the model in a variety of locations and burial environments. To develop the model, giant ragweed seedlings were counted and removed weekly each growing season from 2000 to 2003 in a fallow field located in west central Ohio. Weather data, soil characteristics and geographic location were used to predict soil thermal and moisture conditions with the Soil Temperature and Moisture Model (STM2). Hydrothermal time (θHT) initiated March 1 and base values were extrapolated from the literature (Tb= 2 C, ψb= −10 MPa). Cumulative percent emergence initially increased rapidly and reached 60% of maximum by late April (approximately 400 θHT), leveled off for a period in May, and increased again at a lower rate before concluding in late July (approximately 2,300 θHT). The period in May when few seedlings emerged was not subject to soil temperatures or water potentials less than the θHTbase values. The biphasic pattern of emergence was modeled with two successive Weibull models that were validated in 2005 in a tilled and a no-tillage environment and in 2006 at a separate location in a no-tillage environment. Root-mean-square values for comparing actual and model predicted cumulative emergence values ranged from 8.0 to 9.5%, indicating a high degree of accuracy. This experiment demonstrated an approach to emergence modeling that can be used to forecast emergence on a local basis according to weed biotype and easily obtainable soil and weather data.

We report a novel peak sorting method for the two-dimensional gas chromatography/time-of-flight mass spectrometry (GC x GC/TOF-MS) system. The objective of peak sorting is to recognize peaks from the same metabolite occurring in different samples from thousands of peaks detected in the analytical procedure. The developed algorithm is based on the fact that the chromatographic peaks for a given analyte have similar retention times in all of the chromatograms. Raw instrument data are first processed by ChromaTOF (Leco) software to provide the peak tables. Our algorithm achieves peak sorting by utilizing the first- and second-dimension retention times in the peak tables and the mass spectra generated during the process of electron impact ionization. The algorithm searches the peak tables for the peaks generated by the same type of metabolite using several search criteria. Our software also includes options to eliminate non-target peaks from the sorting results, e.g., peaks of contaminants. The developed software package has been tested using a mixture of standard metabolites and another mixture of standard metabolites spiked into human serum. Manual validation demonstrates high accuracy of peak sorting with this algorithm.

Giant ragweed is a competitive, allergenic weed that persists in agricultural fields and early successional sites. Field experiments were conducted to determine the effects of seed size and seed burial depth on giant ragweed emergence and seed demise. In a seedling emergence experiment, small (< 4.8 mm in diameter) and large (> 6.6 mm in diameter) seeds were buried 0, 5, 10, and 20 cm in fall 1997, and weed emergence was monitored over the next seven growing seasons. A generalized linear mixed model fit to the cumulative emergence data showed that maximum emergence for both seed sizes occurred at the 5-cm burial depth, where probability of emergence was 19% for small seeds and 49% for large seeds. Emergence probability at the 10-cm burial depth was 9% for small seeds and 30% for large seeds, and no seedlings emerged from the 20-cm burial depth. The model predicted that ≥ 98% of total cumulative emergence was completed after four growing seasons for large seeds buried 5 cm, five growing seasons for small seeds buried 5 cm and large seeds buried 10 cm, and seven growing seasons for small seeds buried 10 cm. Seed size and burial treatment effects on seed demise were tested in a second experiment using seed packets. Rates of seed demise were inversely proportional to burial depth, and the percentage of viable seeds remaining after 4 yr ranged from 0% on the soil surface to 19% at the 20-cm burial depth. Some seeds recovered from the 20-cm burial depth were viable after 9 yr of burial. These results, coupled with previous research, suggest that seed size polymorphism facilitates giant ragweed adaptation across habitats and that a combination of no-tillage cropping practices, habitat modification, and timely weed control measures can reduce its active seed bank in agricultural fields by 90% or more after 4 yr.

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was

Studies were conducted from 2001 through 2003 to determine the extent of resistance to acetolactate synthase (ALS) inhibitors and glyphosate in Ohio horseweed biotypes. The response of 66 horseweed biotypes to cloransulam-methyl and glyphosate was determined in the greenhouse. Application of 0.07 kg ai cloransulam/ha reduced plant biomass by less than 60% for 38 of the 66 biotypes. Application of 3.4 kg ae glyphosate/ha reduced biomass by at least 80% for the 51 biotypes collected in 2001, but biomass was similar to that of nontreated plants for 11 of the 15 populations collected in 2002. A dose–response study was conducted with selected biotypes, and a nonlinear, logistic dose–response curve was fit to the data to calculate the herbicide dose required to reduce fresh weight 50% (GR50). On the basis of GR50values, the resistance ratio (R/S) for two ALS-resistant biotypes was 34 and 943 for chlorimuron-ethyl and 32 and 168 for cloransulam, respectively. The R/S ratio for two glyphosate-resistant biotypes was 33 and 39. Results of these studies indicate that, in 2002, ALS-resistant horseweed was widespread throughout Ohio, whereas resistance to glyphosate occurred primarily in several counties in southwestern Ohio.

Glutathione S-transferases (GSTs) are involved in many stress responses in plants, for example, participating in the detoxification of xenobiotics and limiting oxidative damage. Studies examining the regulation of this gene family in diverse plant species have focused primarily on RNA expression. A proteomics method was developed to identify GSTs expressed in Arabidopsis seedlings and to determine how the abundance of these proteins changed in response to copper, a promoter of oxidative stress, and benoxacor, a herbicide safener. Eight GSTs were identified in seedlings grown under control conditions, and only one, AtGSTU19, was induced by benoxacor. In contrast, four GSTs, AtGSTF2, AtGSTF6, AtGSTF7, and AtGSTU19, were significantly more abundant in copper-treated seedlings. The different responses to these treatments may reflect the potential for copper to affect many more aspects of plant growth and physiology compared with a herbicide safener. Differences between RNA and protein expression of GSTs indicate that both transcriptional and translational mechanisms are involved in regulation of GSTs under these conditions.

Giant ragweed seeds have high nutritional value, consisting of 47% crude protein and 38% crude fat, and may be an important food source for rodent and invertebrate populations in agricultural and early successional ecosystems. We investigated temporal patterns of postdispersal giant ragweed seed predation on the soil surface of a no-tillage cornfield as affected by involucre (seed dispersal unit) size and presence or absence of crop residue. Cage exclusion experiments indicated that rodents and invertebrates were the principal predators of giant ragweed seed, and total predation of involucres over a 12-mo period beginning in November was 88%. Rodents were the greatest predators of giant ragweed involucres during fall and winter, and cumulative predation by February 1 in treatments with rodent access ranged from 39 to 43%. In contrast, giant ragweed involucre predation by invertebrates occurred mainly from May 1 to November 1. When rodent access to involucres was prevented, total involucre predation by invertebrates over a 12-mo period ranged from 57 to 78%. Rodents showed an initial preference for large involucres (> 4.8-mm diameter), and invertebrates preferred small involucres (< 4.8-mm diameter). Involucres covered with corn plant residue underwent less predation by rodents from November to February than uncovered involucres, but residue cover had no effect on seed predation by invertebrates. In a laboratory feeding trial, the carabidHarpalus pensylvanicuspreferred seed of smooth pigweed and yellow foxtail to giant ragweed seed, suggesting that giant ragweed seed is an incidental rather than a preferred food source for some carabids. Because giant ragweed exhibits relatively low fecundity and short seed bank persistence, results of this study suggest that postdispersal predation may directly reduce giant ragweed recruitment the next year by reducing new seed bank inputs. However, seed losses from predation alone may be insufficient to maintain giant ragweed populations below economic threshold levels in no-tillage cornfields.

Three studies were conducted in 1999 and 2000 to determine whether acetolactate synthase (ALS)–resistant common ragweed and giant ragweed biotypes were present in Ohio. Results of field studies indicated that biotypes of both species had cross-resistance to three chemical families of ALS-inhibiting herbicides. Cloransulam-methyl applied postemergence at 9, 18, and 36 g/ha controlled more than 85% of two susceptible populations of common and giant ragweed 28 d after treatment, whereas less than 35% control of resistant populations was achieved at the same rates. Fomesafen, lactofen, and glyphosate applied alone at the recommended rates provided the most effective control of ALS-resistant common and giant ragweed. Mixtures of cloransulam-methyl with either fomesafen or lactofen did not significantly increase ALS-resistant common and giant ragweed control compared with each diphenylether herbicide used alone. Dose–response bioassays conducted in the greenhouse indicated that susceptible common and ...

Interest in using crop competitiveness as an integrated weed management tool is increasing. Our objective was to describe traits that could be sources of the competitiveness we previously observed in grain sorghum grown in association with shattercane, which is a common annual weed and a close relative of the crop. Such information could aid in developing management practices for cultivated sorghum to improve its competitiveness with weeds. A bioassay was conducted to compare emergence of the crop and the weed in the greenhouse, and vegetative growth was monitored for 31 d in a within-row competition study. Results described a crop that competed well with the weed and other crop plants and agreed with studies showing that relative time of emergence influenced competitiveness. The mechanism by which grain sorghum emerged before the weed was a by-product of domestication that reduced glumes surrounding the wild-type seeds. This could be shown experimentally by hulling shattercane seeds, which then ...

A field study was conducted to determine the effects of giant ragweed emergence time and population density on corn grain yield, giant ragweed seed production, and giant ragweed predispersal seed losses. When weeds and crop emerged concurrently, hyperbolic regression of percent corn yield loss on giant ragweed population densities of 1.7, 6.9, and 13.8 weeds per 10 m2 gave a predicted loss rate of 13.6% for the first weed per 10 m2 in the linear response range at low densities and a maximum yield loss of 90% at high weed densities. Crop yield loss response to weed density was linear when giant ragweed emerged 4 wk after corn, and the regression coefficient indicated a yield loss rate of 1% per unit increase in weed density. A larger proportion of the variation in corn yield loss was explained by weed density (r2 = 0.99) than by weed biomass (r2 = 0.81). There was a positive linear relationship between giant ragweed seed production and weed density at each weed emergence time. When giant ragweed e...

Pure and biculture stands of rye `Wheeler' (Secale cereale L.) and field pea (Pisum sativum L.) were established and killed for mulch in Spring 1996, 1997, and 1998, in Columbus, Ohio. Treatments were five rye to pea proportions, each with a high, medium, and low seeding rate. Their effects on tomato (Lycopersicon esculentum Mill.) growth and yield were compared with those of a weedy check; a tilled, nonweeded check; and a tilled, hand-weeded check. Tomato tissue and soil were sampled for nutrient analysis. Number of leaves, branching, height, leaf area, dry weight, rate of flowering and fruit set, and fruit yield of tomato plants varied directly with the proportion of pea in the cover crop and decreased with reduced cover crop seeding rates. In 1997, yields of tomato were as high as 50 MT·ha–1 in the 1 rye: 3 pea cover crop; yield was poorest in the weedy check (0.02 MT·ha–1 in 1996). Most of the cover-cropped plots produced better yields than did the conventionally weeded check. No consistent relationship between levels of macro-nutrients in tomato leaf and soil samples and the cover crop treatments was established. Spring-sown rye + pea bicultures (with a higher ratio of pea) have a potential for use in tomato production.

A greenhouse study was conducted to determine the effects of sublethal dicamba concentrations in the nutrient media on hydroponically grown tomato plants. Tomato leaf area was the most sensitive vegetative growth parameter measured in response to dicamba concentrations, ranging from 0 to 22 µg/L. Leaf area was reduced 31 and 76%, and specific leaf weights, a relative measure of leaf thickness (g/cm2), increased 26 and 121% after 30-d exposure to dicamba concentrations of 2.2 and 22 µg/L, respectively. In long-term experiments conducted until plants produced first ripe fruit, regression analysis indicated leaf area reductions of 8 and 66% from initial dicamba concentrations of 1 and 10 µg/L, respectively. Reductions in total fruit fresh weight were highly correlated (r = 0.93) with leaf area reductions caused by dicamba. A hyperbolic regression model gave predicted losses in fruit fresh weight per plant of 6% at 1 µg/L dicamba and 73% at 10 µg/L dicamba (r2 = 0.87). Results generally indicated th...

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation

Bioassays using binary mixtures that included a cover crop with known allelopathic potential and a weed species were employed to determine the importance of allelopathy compared to resource competition as interference mechanisms. Responses of weed species germinated with cover crops in a petri dish were measured. Interference between weed and cover crop seedlings was determined in a greenhouse experiment using the additive design, which included partitions to reduce above- and below-ground competition and used capillary mat subirrigation to control moisture and fertilizer availability. Germinating sorghum reduced radicle length of weeds, whereas germinating rye tended to increase weed radicle length. Methods limited above-ground competition, so likely interference mechanisms were below-ground competition and allelopathy. Germination with a cover crop had little effect on germination and shoot length of weeds. Increased density of rye but not of sorghum reduced growth of barnyardgrass seedlings. Reduced number of barnyardgrass leaves in the presence of rye was likely due to allelopathy. Suppression of barnyardgrass dry weight attributed to allelopathic interference by rye was successfully separated and compared to the combined effects of competition and allelopathy.

Field and laboratory studies were conducted to investigate the mechanisms of weed suppression by cover crops. High-performance liquid chromatograph analysis and a seed germination bioassay demonstrated that rye (Secale cereale L.) can be leached of its allelochemicals, redried, and used as an inert control for separating physical suppression from other types of interference. In a field study, rye, crimson clover (Trifolium incarnatum L.), hairy vetch (Vicia villosa Roth.), barley (Hordeum vulgare L.), and a mixture of the four species suppressed the emergence of eastern black nightshade (Solanum ptycanthum Dun.). Crimson clover inhibited the emergence of eastern black nightshade beyond what could be attributed to physical suppression alone. The emergence of yellow foxtail [Setaria glauca (L.) Beauv.] was inhibited by rye and barley but not by the other cover crops or the cover crop mixture.

Greenhouse experiments that used capillary mat subirrigation to maintain constant soil moisture and to sup- ply fertilizer continuously were conducted to evaluate the effects of sorghum or rye residue on early growth of barn- yardgrass and velvetleaf. The separate effects of root residue and of shoot residue were compared to the combined effects of root plus shoot residues and to an uncovered soil control. Residues included as nontoxic controls were leached shoot tissue and poplar excelsior. Shoot residue, leached shoot tissue, and poplar excelsior were surface-applied on an equal light transmittance basis such that mass of poplar excelsior > shoot residue > leached shoot tissue. The presence of rye root residue delayed emergence of barnyardgrass. Surface- applied residues tended to decrease barnyardgrass height, but velvetleaf stem length was greater in treatments with surface residue. Although cover crop shoot residues had little effect on weed growth after 18 d, weed growth decreased in the presence of cover crop root residues and poplar excel- sior. Nomenclature: Barnyardgrass, Echinochloa crus-galli L. #3 ECHCG; velvetleaf, Abutilon theophrasti Medicus # ABUTH; rye, Secale cereale L. 'Wheeler'; sorghum, Sor- ghum bicolor (L.) Moench 'Pioneer Hybrid #8333.' Additional index words. Capillary mat subirrigation, cover crop, alielopathy, physical effects.

Field studies were conducted to determine the ef- fects of cultivated oats planting pattern on early canopy shape and growth of cultivated oats and wild oats, in part to test the assumption of radial plant canopy expansion on which previous theoretical models of crop-weed interference models have been based. Cultivated oats density was kept constant as the pattern rectangularity was varied, and single wild oats plants were centered within each pattern. Individ- ual plant canopies, photographed from above 31 days after emergence (DAE), were radial for wild oats in all crop plant- ing patterns and for cultivated oats planted in triangular and square planting patterns. Canopy radius perpendicular to the crop row axis in rectangular patterns was similar to canopy radius along the same cardinal axis in equidistant patterns, but was reduced along the crop row axis, resulting in a rectangular canopy shape and decreased canopy area in rectangular compared to equidistant patterns. Cultivated oats dry weight and leaf area at crop flowering (64 DAE) also decreased with increasing rectangularity of crop planting pattern. Reductions in cultivated oats growth in rectangular patterns were associated with earlier intraspecific interfer- ence and delayed crop canopy closure in rectangular com- pared to equidistant patterns. Wild oats leaf area and tiller number 64 DAE decreased with more equidistant crop plant- ing patterns, consistent with reduced canopy area 31 DAE and earlier crop canopy closure in equidistant patterns. The data suggest that individual oats canopy expansion during early growth is essentially radial and also support previous theoretical predictions of crop planting pattern effects on weed suppression. Nomenclature: Wild oats, Avena fatua L. #3 AVEFA; oats, Avena sativa L. 'Ogle' and 'Pennvda.'

Studies were conducted in conventional and no-till- age corn in 1990,1991, and 1992 at Wooster, OH, to measure corn yield and velvetleaf seed production in response to density of early and late emerging velvetleaf, and to estimate economic thresholds. The percent reduction in corn yield fit a hyperbolic function over velvetleaf densities from 1 to 30 plants m2. The percent yield loss and velvetleaf seed produc- tion were higher in a warm, wet year (1990) than in a dry (1991) or cold, wet year (1992). The percent corn yield reduc- tion was generally greater in no-tillage than in conventional tillage and from early rather than late emerging velvetleaf. Maximum velvetleaf seed production ranged from about 18,000 seeds m-2 for early emerging weeds in no-tillage in 1990 to 100 seeds m72 for late emerging weeds in no-tillage in 1992. The single year economic threshold for early emerging velvetleaf ranged from 0.40 to 14.0 velvetleaf mi2 in conven- tional tillage and 0.13 to 3.13 in no-tillage. Economic thresh- olds that were predicted using yield goal information deviated from actual thresholds (using actual yields) for a given year by 43 to 30%. Single year economic thresholds were similar in both tillage treatments, but their value for management decisions is questionable due to variation among growing seasons and weed seed production from subthreshold populations. Nomenclature: velvetleaf, Abu- tilon theophrasti Medicus. #3 ABUTH; corn, Zea mays L. 'DK453.' Additional index words. Economic threshold, competition, seed rain, ABUTH.

The study of weed life cycles, reproductive strategies, and the soil seed bank is emphasized in the undergraduate weed science course at Ohio State University as central to an understanding of the survival of weeds in the environment. A laboratory exercise was conducted every spring and fall academic quarter from 1991 to 1993 to demonstrate the effects of long-term cropping and soil disturbance histories on weed seed banks and aboveground weed communities. Five sites with diverse histories of culture were sampled; these included a field cultivated in vegetables under continuous conventional tillage for 59 yr, a field cultivated in field corn under continuous no-tillage for 11 yr, a 24 yr-old turfgrass research farm, a 70 yr-old forest, and a section of the forest border. Students conducted a survey of the weeds growing at the sites and separated and identified seeds from soil samples over a 3-wk period in weekly 2-h laboratory periods. Students wrote reports interpreting the data based on their knowledge of the site histories, weed life cycles, and weed seed production and longevity characteristics. The data were consistent over academic quarters as well as with published research, indicating that the survey and soil sampling techniques provided a reasonably accurate representation of the weed flora and soil seed populations. Weeds found growing at the sites were primarily summer annuals at the vegetable site, and a mix of summer and winter annuals, biennials, and perennials at the remaining sites. Annual weeds dominated the seed banks of all sites with common lambsquarters, pigweed spp., and common purslane being the most commonly found seeds. The presence of most seeds in the soil could be explained by a combination of species seed production and seed longevity characteristics and species abundance in the standing community. Interpretation of the data required students to integrate and apply lecture material and provided an excellent thinking exercise.

Field experiments were established at Columbus and near South Charleston, OH to determine the effects of giant ragweed population density on soybean yield and to characterize the development of giant ragweed grown in 76-cm soybean rows. An economic threshold was calculated for Ohio using a common treatment for giant ragweed control in soybean. A cost of $41/ha was estimated for a farmer to apply 0.56 kg/ha bentazon plus 0.28 kg/ha fomesafen plus COC (1.25% v/v). Assuming a soybean value of $0.22/kg, the cost of control was equivalent to 5.4 and 7.1% of the soybean yield in 1991 and 1992, respectively, which corresponded to the yield loss caused by 0.08 and 0.03 giant ragweed plants/m2. The competitiveness of giant ragweed can be at least partly attributed to its ability to initiate and maintain axillary leaves and branches within the shaded confines of the soybean canopy.

Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinson's disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the "2O " form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H(2)O(2) resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a protein's ability to undergo oxidative modification, and they imply that stimulating the conversion of DJ-1 to the 2O form may be therapeutically beneficial in familial or sporadic PD.

There is potential that the pathological effects of oxidative stress (OS) associated diseases such as diabetes could be ameliorated with antioxidants, but this will require a clearer understanding of the pathway(s) by which proteins are damaged by OS. This study reports the development and use of methods that assess the efficacy of dietary antioxidant supplementation at a mechanistic level. Data reported here evaluate the impact of green tea supplementation on oxidative stress induced post-translational modifications (OSi-PTMs) in plasma proteins of Zucker diabetic fatty (ZDF) rats. The mechanism of antioxidant protection was examined through both the type and amount of OSi-PTMs using mass spectrometry based identification and quantification. Carbonylated proteins in freshly drawn blood samples were derivatized with biotin hydrazide. Proteins thus biotinylated were selected from plasma samples of green tea fed diabetic rats and control animals by avidin affinity chromatography, further fractionated by reversed phase chromatography (RPC); fractions from the RPC column were tryptic digested, and the tryptic digest was fractionated by RPC before being identified by tandem mass spectrometry (MS/MS). Relative quantification of peptides bearing carbonylation sites was achieved for the first time by RPC-MS/MS using selective reaction monitoring (SRM). Seventeen carbonylated peptides were detected and quantified in both control and treated plasma. The relative concentration of eight was dramatically different between control and green tea treated animals. Seven of the OSi-PTM bearing peptides had dropped dramatically in concentration with treatment while one increased, indicating differential regulation of carbonylation by antioxidants. Green tea antioxidants were found to reduce carbonylation of proteins by lipid peroxidation end products most, followed by advanced glycation end products to a slightly lower extent. Direct oxidation of proteins by reactive oxygen species (ROS) was protected the least by green tea.