The role of citizen science in addressing grand challenges in food and agriculture research.

The power of citizen science to contribute to both science and society is gaining increased recognition, particularly in physics and biology. Although there is a long history of public engagement in agriculture and food science, the term 'citizen science' has rarely been applied to these efforts. Similarly, in the emerging field of citizen science, most new citizen science projects do not focus on food or agriculture. Here, we convened thought leaders from a broad range of fields related to citizen science, agriculture, and food science to highlight key opportunities for bridging these overlapping yet disconnected communities/fields and identify ways to leverage their respective strengths. Specifically, we show that (i) citizen science projects are addressing many grand challenges facing our food systems, as outlined by the United States National Institute of Food and Agriculture, as well as broader Sustainable Development Goals set by the United Nations Development Programme, (ii) there exist emerging opportunities and unique challenges for citizen science in agriculture/food research, and (iii) the greatest opportunities for the development of citizen science projects in agriculture and food science will be gained by using the existing infrastructure and tools of Extension programmes and through the engagement of urban communities. Further, we argue there is no better time to foster greater collaboration between these fields given the trend of shrinking Extension programmes, the increasing need to apply innovative solutions to address rising demands on agricultural systems, and the exponential growth of the field of citizen science.

A DNA microarray-based genetic screen for nonhomologous end-joining mutants in Saccharomyces cerevisiae.

We describe a microarray-based screen performed by imposing different genetic selections on thousands of yeast mutants in parallel, representing most genes in the yeast genome. The presence or absence of mutants was detected by oligonucleotide arrays that hybridize to 20-nucleotide "barcodes." We used this method to screen for components of the nonhomologous end-joining (NHEJ) pathway. Known components of the pathway were identified, as well as a gene not previously known to be involved in NHEJ, NEJ1. Nej1 protein interacts with the amino terminus of LIF1/XRCC4, a recently recognized "guardian of the genome" against cancer.

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.

We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

Experimental annotation of the human genome using microarray technology.

The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.

DNA microarrays for expression profiling.

DNA microarrays enable the transcript levels of an entire genome to be measured simultaneously. Recent improvements in array manufacture, sample preparation, and data analysis are shifting emphasis from the technology itself to experimental design and the broader range of biological questions that can be addressed. The past year has also seen a transition from experiments involving a small number of conditions, with an emphasis on the specific genes induced or repressed, to experiments involving hundreds of conditions in which patterns of global gene expression are used to classify disease specimens and discover gene functions and drug targets. Basic research, medicine, and pharmacology are all likely to benefit from these advances.

Wolbachia infections in native and introduced populations of fire ants (Solenopsis spp.).

Wolbachia are cytoplasmically inherited bacteria that induce a variety of effects with fitness consequences on host arthropods, including cytoplasmic incompatibility, parthenogenesis, male-killing and feminization. We report here the presence of Wolbachia in native South American populations of the fire ant Solenopsis invicta, but the apparent absence of the bacteria in introduced populations of this pest species in the USA. The Wolbachia strains in native S. invicta are of two divergent types (A and B), and the frequency of infection varies dramatically between geographical regions and social forms of this host. Survey data reveal that Wolbachia also are found in other native fire ant species within the Solenopsis saevissima species complex from South America, including S. richteri. This latter species also has been introduced in the USA, where it lacks Wolbachia. Sequence data reveal complete phylogenetic concordance between mtDNA haplotype in S. invicta and Wolbachia infection type (A or B). In addition, the mtDNA and associated group A Wolbachia strain in S. invicta are more closely related to the mtDNA and Wolbachia strain found in S. richteri than they are to the mtDNA and associated group B Wolbachia in S. invicta. These data are consistent with historical introgression of S. richteri cytoplasmic elements into S. invicta populations, resulting in enhanced infection and mtDNA polymorphisms in S. invicta. Wolbachia may have significant fitness effects on these hosts (either directly or by cytoplasmic incompatibility) and therefore these microbes potentially could be used in biological control programmes to suppress introduced fire ant populations.

Functional discovery via a compendium of expression profiles.

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Assessing genetic structure with multiple classes of molecular markers: a case study involving the introduced fire ant Solenopsis invicta.

We used 30 genetic markers of 6 different classes to describe hierarchical genetic structure in introduced populations of the fire ant Solenopsis invicta. These included four classes of presumably neutral nuclear loci (allozymes, codominant random amplified polymorphic DNAs (RAPDs), microsatellites, and dominant RAPDs), a class comprising two linked protein-coding nuclear loci under selection, and a marker of the mitochondrial DNA (mtDNA). Patterns of structure revealed by F statistics and exact tests of differentiation were highly concordant among the four classes of neutral nuclear markers, although the microsatellites were the most effective markers for detecting structure. The results from the mtDNA complemented those from the neutral nuclear markers by revealing that strong limitations to female-mediated gene flow were the cause of the local structure registered by the nuclear markers. The pattern of structure inferred from the selected nuclear loci was markedly different from the patterns derived from the other sets of markers but was predictable on the basis of the presumed mode of selection acting on these loci. In general, the results for all six classes of markers can be explained by known features of the social and reproductive biology of fire ants. Thus, the results from these diverse sets of markers, combined with detailed natural history data, provide an unusually complete picture of how the fundamental evolutionary forces of gene flow, drift, and selection govern the distribution of genetic variation within and between fire ant populations.

Genomic profiling of drug sensitivities via induced haploinsufficiency.

Lowering the dosage of a single gene from two copies to one copy in diploid yeast results in a heterozygote that is sensitized to any drug that acts on the product of this gene. This haploinsufficient phenotype thereby identifies the gene product of the heterozygous locus as the likely drug target. We exploited this finding in a genomic approach to drug-target identification. Genome sequence information was used to generate molecularly tagged heterozygous yeast strains that were pooled, grown competitively in drug and analysed for drug sensitivity using high-density oligonucleotide arrays. Individual heterozygous strain analysis verified six known drug targets. Parallel analysis identified the known target and two hypersensitive loci in a mixed culture of 233 strains in the presence of the drug tunicamycin. Our discovery that both drug target and hypersensitive loci exhibit drug-induced haploinsufficiency may have important consequences in pharmacogenomics and variable drug toxicity observed in human populations.

Hierarchical analysis of genetic structure in native fire ant populations: results from three classes of molecular markers.

We describe genetic structure at various scales in native populations of the fire ant Solenopsis invicta using two classes of nuclear markers, allozymes and microsatellites, and markers of the mitochondrial genome. Strong structure was found at the nest level in both the monogyne (single queen) and polygyne (multiple queen) social forms using allozymes. Weak but significant microgeographic structure was detected above the nest level in polygyne populations but not in monogyne populations using both classes of nuclear markers. Pronounced mitochondrial DNA (mtDNA) differentiation was evident also at this level in the polygyne form only. These microgeographic patterns are expected because polygyny in ants is associated with restricted local gene flow due mainly to limited vagility of queens. Weak but significant nuclear differentiation was detected between sympatric social forms, and strong mtDNA differentiation also was found at this level. Thus, queens of each form seem unable to establish themselves in nests of the alternate type, and some degree of assortative mating by form may exist as well. Strong differentiation was found between the two study regions using all three sets of markers. Phylogeographic analyses of the mtDNA suggest that recent limitations on gene flow rather than longstanding barriers to dispersal are responsible for this large-scale structure.

Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy.

A quantitative and highly parallel method for analysing deletion mutants has been developed to aid in determining the biological function of thousands of newly identified open reading frames (ORFs) in Saccharomyces cerevisiae. This approach uses a PCR targeting strategy to generate large numbers of deletion strains. Each deletion strain is labelled with a unique 20-base tag sequence that can be detected by hybridization to a high-density oligonucleotide array. The tags serve as unique identifiers (molecular bar codes) that allow analysis of large numbers of deletion strains simultaneously through selective growth conditions. Hybridization experiments show that the arrays are specific, sensitive and quantitative. A pilot study with 11 known yeast genes suggests that the method can be extended to include all of the ORFs in the yeast genome, allowing whole genome analysis with a single selective growth condition and a single hybridization.

Mobilization of Ca2+ influx, but not of stored Ca2+, by extracellular ATP in rat submandibular gland acini.

To determine its effect on cytosolic Ca2+ levels (Ca2+i) and to assess its role as an autonomic cotransmitter, ATP was added to suspensions of rat pancreatic or submandibular gland acini loaded with fura-2. ATP had no effect on pancreatic acinar Ca2+i at either high (2.5 mM) or low (< 10 nM) extracellular Ca2+ concentrations (Ca2+o). In submandibular acini, ATP had little effect on Ca2+i when Ca2+o was < 10 nM but, with Ca2+o equal to 2.5 mM, ATP increased Ca2+i to a steady-state level 2-4 times the resting level of 50-100 nM. Addition of carbachol or epinephrine further increased Ca2+i. The mean and standard deviation of the ATP4- concentration at half of maximal activity (K0.5 ATP4-) was 33 +/- 7 microM and the Hill coefficient was 1.5 +/- 0.7 (n = 9). The active species is apparently ATP4-, as adding Mg2+ increases the total concentration of ATP needed to elevate Ca2+i. GTP, UTP, ADP and adenosine produced no significant changes in Ca2+i. However, 3'-O-(4-benzoyl)benzoyl ATP (BZATP) increased Ca2+i with a K0.5 BZATP of 2.4 +/- 0.5 microM and a Hill coefficient of 2.8 +/- 0.4 (n = 8). ATP-induced changes in Ca2+i were not due to a generalized increase in acinar-cell permeability and were unaffected by voltage-dependent Ca2+ channel blockers or by the presence of depolarizing concentrations of K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular ATP prevents the release of stored Ca2+ by autonomic agonists in rat submandibular gland acini.

In dose-dependent fashion, extracellular ATP reduces the increase in cytosolic Ca2+ concentration ([Ca2+]o) due to mobilization of cellular Ca2+ stores by both epinephrine [half-maximal inhibitory concentration (IC50) = 35.7 +/- 12.9 microM; Hill coefficient (NH) = -2.0 +/- 0.7, n = 8] and by carbachol (IC50 = 27.0 +/- 7.0 microM, NH = -2.3 +/- 0.7, n = 9). Inhibition is due to ATP4- but does not result from any emptying or inaccessibility of Ca2+ stores, which are readily mobilized by thapsigargin in the presence of ATP4-. Reduction of Ca2+ mobilization is rapid but is not due to direct interference by ATP with the interaction of carbachol or epinephrine with their respective cell surface receptors. A benzoyl derivative of ATP, 3'-O-(4-benzoyl) adenosine 5'-triphosphate (BZATP) is more potent than ATP in reducing [Ca2+]i due to mobilization of stored Ca2+ by either carbachol or epinephrine (IC50 for carbachol = 3.9 +/- 0.4 microM, NH = -3.2 +/- 0.5; IC50 for epinephrine = 3.8 +/- 0.2, NH = -2.6 +/- 0.7, n = 3) but GTP, UTP, ADP, and adenosine do not inhibit mobilization of stored Ca2+ by either carbachol or epinephrine. Neither ATP nor BZATP prevents the influx of extracellular Ca2+ stimulated by carbachol or epinephrine. These results suggest that ATP inhibits Ca2+ mobilization by autonomic neurotransmitters after occupation of P2Z purinoceptors.

High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus.

A thermostable DNA polymerase which possesses an associated 3'-to-5' exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10% of the number of mutations obtained from similar amplifications performed with Taq DNA polymerase. The PCR fidelity assay is based on the amplification and cloning of lacI, lacO and lacZ alpha gene sequences (lacIOZ alpha) using either Pfu or Taq DNA polymerase. Certain mutations within the lacI gene inactivate the Lac repressor protein and permit the expression of beta Gal. When plated on a chromogenic substrate, these LacI- mutants exhibit a blue-plaque phenotype. These studies demonstrate that the error rate per nucleotide induced in the 182 known detectable sites of the lacI gene was 1.6 x 10(-6) for Pfu DNA polymerase, a greater than tenfold improvement over the 2.0 x 10(-5) error rate for Taq DNA polymerase, after approx. 10(5)-fold amplification.

Structural and functional analysis of EcoRI DNA methyltransferase by proteolysis.

Native EcoRI DNA methyltransferase (Mtase, Mr 38,050) is proteolyzed by trypsin to generate an intermediate 36-kDa fragment (p36) followed by the formation of two polypeptides of Mr 23,000 and 13,000 (p23 and p13, respectively). Protein sequence analysis of the tryptic fragments indicates that p36 results from removal of the first 14 or 16 amino acids, p23 spans residues 15-216, and p13 spans residues 217-325. The relative resistance to further degradation of p23 and p13 suggests stable domain structures. This is further supported by the generation of similar fragments with SV8 endoprotease which has entirely different peptide specificities. Our results suggest the Mtase is a two-domain protein connected by a highly flexible interdomain hinge. The putative hinge region encompasses previously identified peptides implicated in AdoMet binding [Reich, N.O., & Everett, E. (1990) J. Biol. Chem. 265, 8929-8934] and catalysis [Everett et al. (1990) J. Biol. Chem. 265, 17713-17719]. Protection studies with DNA, S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy), and sinefungin (AdoMet analogue) show that the Mtase undergoes significant conformational changes upon ligand binding. Trypsinolysis of the AdoMet-bound form of the Mtase generates different fragments, and the AdoMet-bound form is over 800 times more stable than unbound Mtase. The sequence-specific ternary complex (Mtase-DNA-sinefungin) is 2000 times more resistant to degradation by trypsin; cleavage eventually generates 26- and 12-kDa fragments which span residues 104-325 and 1-103, respectively (p26 and p12). The first 14 or 16 amino acids of the Mtase are not essential since p36 retains activity. Activity analysis of the p26 and p12 mixture also indicates retention of activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical toxicity associated with tiazofurin.

Tiazofurin, an investigational antimetabolite, is undergoing clinical evaluation in leukemia. We analyzed the data base of 198 patients entered in Phase I trials to characterize the incidence and severity of toxicities associated with tiazofurin according to dose and schedule. Severe myelosuppression occurred infrequently, and was not dose-dependent. A five day bolus schedule had a higher incidence of severe or life-threatening neutropenia than other schedules. Tiazofurin produced lymphopenia which was not dose-dependent in the range of 23-36% decrease from baseline, and the effect on lymphocyte count was generally greater than the decline in neutrophil count. Non-hematologic toxicity of a moderate or worse severity (greater than or equal to grade 2) included nausea and vomiting (18% of all courses), serum transaminase elevations (SGOT, 16%; SGPT, 9%), rash (9%), stomatitis (3%), conjunctivitis (3%), headache (10%), other signs of central nervous system toxicity (8%), and cardiac toxicity, primarily pleuropericarditis (4%). Dose-related cutaneous toxicity, headache, and nausea and vomiting were evident in the five day bolus schedule, and myalgia was more frequently reported at higher doses on the single dose schedule. The five day continuous infusion (CI) schedule had a higher incidence of neurotoxicity, cardiac toxicity, SGPT elevations and ocular toxicity than the daily for five days bolus schedule, but none of these differences attained statistical significance. Although the peak plasma concentrations of tiazofurin achieved with the five day bolus schedule were 3-fold higher than the steady-state plasma levels seen with an equal dose given by CI, the area under the concentration-time curve (AUC) was approximately 1.6-fold higher with CI. These observations suggest that both high peak plasma concentrations (above 400 microM) and prolonged exposure to plasma levels exceeding 50 microM may result in a higher incidence of serious non-hematologic toxicity.

Correlates of severe or life-threatening toxic effects from trimetrexate.

Trimetrexate, an investigational antifol, has been associated with marked variability in drug tolerance among patients. The agent is extensively protein bound, and hepatic biotransformation plays a major role in its elimination. In early phase II testing, nine of 15 patients who experienced life-threatening or fatal toxic effects from trimetrexate had albumin levels less than or equal to 3.5 g/dL prior to treatment. This prompted a review of the data base on 272 patients entered in phase I clinical trails. The incidence of severe or life-threatening anemia, leukopenia, neutropenia, thrombocytopenia, mucositis, and hepatic toxic effects during the first course of trimetrexate was analyzed according to dose, schedule, prior treatment, and baseline protein and albumin levels. The schedules using doses given by short infusions of 30-60 minutes daily for 5 days or weekly for 3 weeks were generally associated with higher incidence of toxic effects than the schedules using doses given every other week by short infusions or those using continuous infusion. The occurrence of leukopenia and mucositis was dose related. Patients with baseline albumin levels less than or equal to 3.5 g/dL had higher incidence of all types of severe or life-threatening toxic effects than those with albumin levels greater than or equal to 3.6 g/dL, and the differences were significant for the development of anemia, thrombocytopenia, and mucositis. Similar correlations were noted for pretreatment protein levels less than or equal to 6.0 g/dL. The small cohort of patients with leukemia experienced substantial toxic effects and tended to have low protein and albumin levels. Performance status and prior therapy did not emerge as strong predictors of severe toxic effects in the univariate analysis. Multivariate analysis confirmed that the type of cancer (leukemia vs. solid tumor), dose, schedule, and baseline albumin level were significant and independent predictors of severe and life-threatening toxic effects in the phase I patient population. Multivariate analysis including only patients with solid tumors indicated that albumin level, dose, and schedule remained significant predictors of toxic effects. Since normal liver function as reflected by bilirubin and transaminase values were a requirement for eligibility, the results suggest that albumin and protein levels may provide a more sensitive index of hepatic function. Patients with hypoalbuminemia and hypoproteinemia are at increased risk of experiencing severe or life-threatening toxic effects from trimetrexate and should be treated cautiously.

Arabinosyl-5-azacytosine: a novel nucleoside entering clinical trials.

Arabinosyl-5-azacytosine is a new compound which has been selected by the Division of Cancer Treatment, National Cancer Institute for clinical development as an antineoplastic agent based on its high degree of activity against a broad range of tumor types in preclinical studies. Therapeutic activity has been observed against murine and human leukemias, transplantable murine solid tumors, and human tumor xenografts. Arabinosyl-5-azacytosine exhibited a broader spectrum of activity against human solid tumors than cytosine arabinoside. Arabinosyl-5-azacytosine is phosphorylated to the nucleotide level by deoxycytidine kinase. Upon further anabolism to the triphosphate level, it can be incorporated into DNA. The mechanism of cytotoxicity is thought to be related to inhibition of DNA synthesis. Leukemic and solid tumor cell lines that are resistant to cytosine arabinoside due to deletion of deoxycytidine kinase activity are cross-resistant to arabinosyl-5-azacytosine. Unlike cytosine arabinoside, arabinosyl-5-azacytosine does not readily undergo deamination. Schedule dependence has been demonstrated in mice bearing L1210 leukemia, with superior activity seen with multiple doses administered on each treatment day compared to administration of larger but less frequently administered doses. From preliminary data in solid tumor models, however, antitumor activity did not appear to be superior with continuous infusion compared to that observed on a bolus schedule. Preclinical toxicology studies indicated that the bone marrow and gastrointestinal tract were the main target organs. A single large dose of arabinosyl-5-azacytosine could be tolerated by both mice and dogs. When administered as a continuous infusion, the toxicity was related to both the dose and duration of exposure, suggesting that toxicity resulted from a critical time above a threshold concentration as opposed to the total area under the concentration-time curve. Phase I clinical trials have been initiated to determine the maximum tolerated dose on a low dose continuous infusion schedule for 72 hours and also on a high dose short infusion daily times five schedule.