Alpha-synuclein ferrireductase activity is detectible in vivo, is altered in Parkinson's disease and increases the neurotoxicity of DOPAL.

The normal cellular role of α-synuclein is of potential importance in understanding diseases in which an aggregated form of the protein has been implicated. A potential loss or change in the normal function of α-synuclein could play a role in the aetiology of diseases such as Parkinson's disease. Recently, it has been suggested that α-synuclein could cause the enzymatic reduction of iron and a cellular increase in Fe(II) levels. Experiments were carried out to determine if such activity could be measured in vivo. Experiments with rats overexpressing human α-synuclein in nigral dopaminergic neurons demonstrated a correlation between α-synuclein expression and ferrireductase activity. Furthermore, studies on tissue from Parkinson's disease patient brains showed a significant decrease in ferrireductase activity, possibly due to deposition of large amounts of inactive protein. Cellular studies suggest that increase ferrireductase activity results in increased levels of dopamine metabolites and increased sensitivity to the toxicity of DOPAL. These findings demonstrate that α-synuclein ferrireductase activity is present in vivo and its alteration may play a role in neuron loss in disease.

Fabrication and evaluation of a micro(bio)sensor array chip for multiple parallel measurements of important cell biomarkers.

This report describes the design and development of an integrated electrochemical cell culture monitoring system, based on enzyme-biosensors and chemical sensors, for monitoring indicators of mammalian cell metabolic status. MEMS technology was used to fabricate a microwell-format silicon platform including a thermometer, onto which chemical sensors (pH, O2) and screen-printed biosensors (glucose, lactate), were grafted/deposited. Microwells were formed over the fabricated sensors to give 5-well sensor strips which were interfaced with a multipotentiostat via a bespoke connector box interface. The operation of each sensor/biosensor type was examined individually, and examples of operating devices in five microwells in parallel, in either potentiometric (pH sensing) or amperometric (glucose biosensing) mode are shown. The performance characteristics of the sensors/biosensors indicate that the system could readily be applied to cell culture/toxicity studies.

Development of an amperometric screen-printed galactose biosensor for serum analysis.

The development of a disposable amperometric biosensor for the measurement of circulating galactose in serum is described. The biosensor comprises a screen-printed carbon electrode (SPCE), incorporating the electrocatalyst cobalt phthalocyanine (CoPC), which is covered by a permselective cellulose acetate (CA) membrane and a layer of immobilized galactose oxidase (GALOX). The optimal response of the biosensor, designated as GALOX-CA-CoPC-SPCE, was obtained by systematically examining the effects of enzyme loading, temperature, pH, and buffer strength. The optimal performance of the biosensor occurred with 2U of GALOX, at 35°C, using 50mM phosphate buffer solution (pH 7.0). The sensitivity was 7.00µAmM(-1)cm(-2) and the linear range from 0.1 to 25mM with a calculated limit of detection (LOD) of 0.02mM; this concentration range and LOD are appropriate to diagnose galactosemia, i.e., concentrations >1.1mM in infants. When the biosensor was used in conjunction with amperometry in stirred solution for the analysis of serum, the precision values obtained on unspiked (endogenous level of 0.153mM) and spiked serum (1mM added) (n=6) were 1.10% and 0.11%, respectively, with a calculated recovery of 99.9%.

Use of whole blood for analysis of disease-associated biomarkers.

Analyses for diagnosis and monitoring of pathological conditions often rely on blood samples, partly due to relative ease of collection. However, many interfering substances largely preclude the use of whole blood itself, necessitating separation of plasma or serum. We present a feasibility study demonstrating potential use of fresh or frozen whole blood to detect soluble biomarkers using an enzyme-linked immunosorbent assay (ELISA)-based method. Good correlation between levels of soluble CD25 in plasma and whole blood of healthy individuals or Alzheimer's patients was established. These results provide a basis for development of a novel biosensor approach for disease-associated biomarker detection in whole blood.

A series of eIF4E alleles at the Bc-3 locus are associated with recessive resistance to Clover yellow vein virus in common bean.

Clover yellow vein virus (ClYVV) is capable of causing severe damage to common bean (Phaseolus vulgaris L.) production worldwide. The snap bean market class is particularly vulnerable because infection may lead to distortion and necrosis of the fresh green pods and rejection of the harvest. Three putatively independent recessive genes (cyv, desc, bc-3) have been reported to condition resistance to ClYVV; however, their allelic relationships have not been resolved. We identified, evaluated, and characterized the phenotypic and molecular genetic variation present in 21 informative common bean genotypes for resistance to ClYVV. Allelism testing phenotypes from multiple populations provided clear evidence that the three genes were a series of recessive alleles at the Bc-3 locus that condition unique potyvirus strain- and species-specific resistance spectra. Candidate gene analysis revealed complete association between the recessive resistance alleles and unique patterns of predicted amino acid substitutions in P. vulgaris eukaryotic translation initiation factor 4E (PveIF4E). This led to the discovery and characterization of two novel PveIF4E alleles associated with resistance to ClYVV, PveIF4E (3) , and PveIF4E (4) . We developed KASPar allele-specific SNP genotyping assays and demonstrated their ability to accurately detect and differentiate all of the PveIF4E haplotypes present in the germplasm, allelism testing, and in three separate segregating populations. The results contribute to an enhanced understanding and accessibility of the important potyvirus resistance conditioned by recessive alleles at Bc-3. The KASPar assays should be useful to further enable germplasm exploration, allelic discrimination, and marker-assisted introgression of bc-3 alleles in common bean.

Human and dog Bayesian dietary mixing models using bone collagen stable isotope ratios from ancestral Iroquoian sites in southern Ontario.

Under the archaeological canine surrogacy approach (CSA) it is assumed that because dogs were reliant on humans for food, they had similar diets to the people with whom they lived. As a result, the stable isotope ratios of their tissues (bone collagen and apatite, tooth enamel and dentine collagen) will be close to those of the humans with whom they cohabited. Therefore, in the absence of human tissue, dog tissue isotopes can be used to help reconstruct past human diets. Here δ13C and δ15N ratios on previously published dog and human bone collagen from fourteenth-seventeenth century AD ancestral Iroquoian village archaeological sites and ossuaries in southern Ontario are used with MixSIAR, a Bayesian dietary mixing model, to determine if the dog stable isotope ratios are good proxies for human isotope ratios in dietary modeling for this context. The modeling results indicate that human dietary protein came primarily from maize and high trophic level fish and dogs from maize, terrestrial animals, low trophic level fish, and human feces. While isotopes from dog tissues can be used as general analogs for human tissue isotopes under CSA, greater insights into dog diets can be achieved with Bayesian dietary mixing models.

Genome-Wide Association Study to Identify Possible Candidate Genes of Snap Bean Leaf and Pod Color.

Color can be an indicator of plant health, quality, and productivity, and is useful to researchers to understand plant nutritional content in their studies. Color may be related to chlorophyll content and photosynthetic activity and provides information for those studying diseases and mineral nutrition because every nutrient deficiency and many diseases produce symptoms that affect color. In order to identify significant loci related to both leaf and pod color in a snap bean (Phaseolus vulgaris L.) diversity panel, a genome-wide association study (GWAS) was carried out. Leaf color in one and pod traits in multiple environments were characterized using a colorimeter. L*a*b* color data were recorded and used to calculate chroma (C*) and hue angle (H°). Leaves were evaluated at three positions (lower, middle, and upper) in the canopy and both pod exterior and interior colors were obtained. GWAS was conducted using two reference genomes that represent the Andean (G19833) and Middle American (5-593) domestication centers. Narrow sense heritabilities were calculated using the mixed linear model (MLM) method in genome association and prediction integrated tool (GAPIT), and significant single nucleotide polymorphisms (SNPs) for each color parameter were obtained using the Bayesian-information and linkage-disequilibrium iteratively nested keyway (BLINK) GWAS model with two principal components (PCAs). In comparison to pod color traits, narrow sense heritabilities of leaf traits were low and similar for both reference genomes. Generally, narrow sense heritability for all traits was highest in the lower, followed by middle, and then upper leaf positions. Heritability for both pod interior and exterior color traits was higher using the G19833 reference genome compared to 5-593 when evaluated by year and means across years. Forty-five significant SNPs associated with leaf traits and 872 associated with pods, totaling 917 significant SNPs were identified. Only one SNP was found in common for both leaf and pod traits on Pv03 in the 5-593 reference genome. One-hundred thirteen significant SNPs, 30 in leaves and 83 in pods had phenotypic variation explained (PVE) of 10% or greater. Fourteen SNPs (four from G19833 and ten from 5-593) with ≥10 PVE%, large SNP effect, and largest p-value for L* and H° pod exterior was identified on Pv01, Pv02, Pv03, and Pv08. More SNPs were associated with pod traits than with leaf traits. The pod interior did not exhibit colors produced by anthocyanins or flavonols which allowed the differentiation of potential candidate genes associated with chloroplast and photosynthetic activity compared to the pod exterior where candidate genes related to both flavonoids and photosynthesis affected color. Several SNPs were associated with known qualitative genes including the wax pod locus (y), persistent color (pc), purple pods (V), and two genes expressed in seeds but not previously reported to affect other plant tissues (B and J). An evaluation of significant SNPs within annotated genes found a number, within a 200 kb window, involved in both flavonoid and photosynthetic biosynthetic pathways.

Genetic variation in a tepary bean (Phaseolus acutifolius A. Gray) diversity panel reveals loci associated with biotic stress resistance.

Tepary bean (Phaseolus acutifolius A. Gray), indigenous to the arid climates of northern Mexico and the Southwest United States, diverged from common bean (Phaseolus vulgaris L.), approximately 2 million years ago and exhibits a wide range of resistance to biotic stressors. The tepary genome is highly syntenic to the common bean genome providing a foundation for discovery and breeding of agronomic traits between these two crop species. Although a limited number of adaptive traits from tepary bean have been introgressed into common bean, hybridization barriers between these two species required the development of bridging lines to alleviate this barrier. Thus, to fully utilize the extant tepary bean germplasm as both a crop and as a donor of adaptive traits, we developed a diversity panel of 422 cultivated, weedy, and wild tepary bean accessions which were then genotyped and phenotyped to enable population genetic analyses and genome-wide association studies for their response to a range of biotic stressors. Population structure analyses of the panel revealed eight subpopulations and the differentiation of botanical varieties within P. acutifolius. Genome-wide association studies revealed loci and candidate genes underlying biotic stress resistance including quantitative trait loci for resistance to weevils, common bacterial blight, Fusarium wilt, and bean common mosaic necrosis virus that can be harnessed not only for tepary bean but also common bean improvement.

A robust SNP-haplotype assay for Bct gene region conferring resistance to beet curly top virus in common bean (Phaseolus vulgaris L.).

Beet curly top virus (BCTV), which is synonymous with curly top virus (CTV), causes significant yield loss in common bean (snap and dry beans) cultivars and several other important crops. Common bean cultivars have been found to be resistant to CTV, but screening for resistance is challenging due to the cyclical nature of epidemics and spotty feeding by the leafhopper that vectors the virus. We used an SNP dataset for the Snap Bean Association Panel (SnAP) agro-inoculated with CTV-Logan (CA/Logan) strain to locate the Bct gene region to a 1.7-Mb interval on chromosome Pv07 using genome-wide association study (GWAS) analysis. Recombinant lines from the SnAP were used to further narrow the Bct region to a 58.0-kb interval. A missense SNP (S07_2970381) in candidate gene Phvul.007G036300 Exonuclease V (EXO5) was identified as the most likely causal mutation, and it was the most significant SNP detected by GWAS in a dry bean population (DBP) naturally infected by the CTV-Worland (Wor) strain. Tm-shift assay markers developed for SNP S07_2970381 and two linked SNPs, S07_2970276 and S07_2966197, were useful for tracking different origins of the Bct EXO5 candidate gene resistance to CTV in common bean. The three SNPs identified four haplotypes, with haplotype 3-1 (Haplo3-1) of Middle American origin associated with the highest levels of CTV resistance. This SNP-haplotype assay will enable breeders to track resistance sources and to develop cultivars with better CTV resistance.

Mapping of the bs5 and bs6 non-race-specific recessive resistances against bacterial spot of pepper.

Bacterial spot caused by Xanthomonas euvesicatoria is a major disease of pepper (Capsicum annuum L.) in warm and humid production environments. Use of genetically resistant cultivars is an effective approach to manage bacterial spot. Two recessive resistance genes, bs5 and bs6, confer non-race-specific resistance against bacterial spot. The objective of our study was to map these two loci in the pepper genome. We used a genotyping-by-sequencing approach to initially map the position of the two resistances. Segregating populations for bs5 and bs6 were developed by crossing susceptible Early CalWonder (ECW) with near-isogenic lines ECW50R (bs5 introgression) or ECW60R (bs6 introgression). Following fine-mapping, bs5 was delimited to a ~535 Kbp interval on chromosome 3, and bs6 to a ~666 Kbp interval in chromosome 6. We identified 14 and 8 candidate resistance genes for bs5 and bs6, respectively, based on predicted protein coding polymorphisms between ECW and the corresponding resistant parent. This research enhances marker-assisted selection of bs5 and bs6 in breeding programs and is a crucial step towards elucidating the molecular mechanisms underlying the resistances.

The Monongahela tradition in "real time": Bayesian analysis of radiocarbon dates.

Despite advances in techniques, methods, and theory, northeastern North American archaeologists continue to use early to mid-twentieth century culture historical taxa as units of analysis and narrative. There is a distinct need to move away from this archaeological practice to enable fuller understandings of past human lives. One tool that enables such a move is Bayesian analysis of radiocarbon dates, which provides a means of constructing continuous chronologies. A large dataset of radiocarbon dates for late prehistoric (ca AD 900/1000-1650) sites in the lower upper Ohio River basin in southwestern Pennsylvania and adjacent portions of Maryland, Ohio, and West Virginia is used here as an example. The results allow a preliminary assessment of how the settlement plans of contemporaneous villages varied considerably, reflecting decisions of the village occupants how to structure built environments to meet their needs.

Genome-Wide Association Study (GWAS) of White Mold Resistance in Snap Bean.

White mold can result in snap bean yield losses of 90 to 100% when field conditions favor the pathogen. A genome-wide association study (GWAS) was conducted to detect loci significantly associated with white mold resistance in a panel of snap bean (Phaseolus vulgaris L.) cultivars. Two populations of snap bean were used in this study. The first population was the BeanCAP (Coordinated Agriculture Project) Snap Bean Diversity Panel (SBDP) (n = 136), and the second population was the Snap Bean Association Panel (SnAP) (n = 378). SBDP was evaluated for white mold reaction in the field in 2012 and 2013, and SnAP was screened in a greenhouse only using the seedling straw test in 2016. Two reference genomes representing the Andean and Middle American centers of domestication were utilized to align the genotyping-by-sequencing (GBS) data. A GWAS was performed using FarmCPU with one principal component after comparing five models. Thirty-four single-nucleotide polymorphisms (SNPs) significantly associated with white mold resistance were detected. Eleven significant SNPs were identified by the seedling straw test, and 23 significant SNPs were identified by field data. Fifteen SNPs were identified within a 100 kb window containing pentatricopeptide repeat (PPR)-encoding genes, and eleven were close to leucine-rich repeat (LRR)-encoding genes, suggesting that these two classes are of outsized importance for snap bean resistance to white mold.

Resolving Indigenous village occupations and social history across the long century of European permanent settlement in Northeastern North America: The Mohawk River Valley ~1450-1635 CE.

The timeframe of Indigenous settlements in Northeast North America in the 15th-17th centuries CE has until very recently been largely described in terms of European material culture and history. An independent chronology was usually absent. Radiocarbon dating has recently begun to change this conventional model radically. The challenge, if an alternative, independent timeframe and history is to be created, is to articulate a high-resolution chronology appropriate and comparable with the lived histories of the Indigenous village settlements of the period. Improving substantially on previous initial work, we report here high-resolution defined chronologies for the three most extensively excavated and iconic ancestral Kanien'kehá꞉ka (Mohawk) village sites in New York (Smith-Pagerie, Klock and Garoga), and a fourth early historic Indigenous site, Brigg's Run, and re-assess the wider chronology of the Mohawk River Valley in the mid-15th to earlier 17th centuries. This new chronology confirms initial suggestions from radiocarbon that a wholesale reappraisal of past assumptions is necessary, since our dates conflict completely with past dates and the previously presumed temporal order of these three iconic sites. In turn, a wider reassessment of northeastern North American early history and re-interpretation of Atlantic connectivities in the later 15th through early 17th centuries is required. Our new closely defined date ranges are achieved employing detailed archival analysis of excavation records to establish the contextual history for radiocarbon-dated samples from each site, tree-ring defined short time series from wood charcoal samples fitted against the radiocarbon calibration curve ('wiggle-matching'), and Bayesian chronological modelling for each of the individual sites integrating all available prior knowledge and radiocarbon dating probabilities. We define (our preferred model) most likely (68.3% highest posterior density) village occupation ranges for Smith-Pagerie of ~1478-1498, Klock of ~1499-1521, Garoga of ~1550-1582, and Brigg's Run of ~1619-1632.

The tepary bean genome provides insight into evolution and domestication under heat stress.

Tepary bean (Phaseolus acutifolis A. Gray), native to the Sonoran Desert, is highly adapted to heat and drought. It is a sister species of common bean (Phaseolus vulgaris L.), the most important legume protein source for direct human consumption, and whose production is threatened by climate change. Here, we report on the tepary genome including exploration of possible mechanisms for resilience to moderate heat stress and a reduced disease resistance gene repertoire, consistent with adaptation to arid and hot environments. Extensive collinearity and shared gene content among these Phaseolus species will facilitate engineering climate adaptation in common bean, a key food security crop, and accelerate tepary bean improvement.

Electrocatalytic behaviour of citric acid at a cobalt phthalocyanine-modified screen-printed carbon electrode and its application in pharmaceutical and food analysis.

Cobalt phthalocyanine-modified screen-printed carbon electrodes (CoPC-SPCEs) have been investigated as disposable sensors for the measurement of citric acid. The analyte was found to undergo an electrocatalytic oxidation process involving the Co(2+)/Co(3+) redox couple. Calibration plots were found to be linear in the range 2 mM to 2.0 M; replicate determinations of a 5.2 mM citric acid (n = 4) solution gave a coefficient of variation of 1.43%. Additions of metal ions, such as Ag(+), Pb(2+), Cu(2+), Fe(3+) and Ca(2+), were found not to interfere. The effects of hesperidin, cysteine, ethylenediaminetetraacetic acid (EDTA), ascorbic, formic, malic, malonic, tartaric, oxalic and trichloroacetic acids on the determination of citric acid were examined and, under the conditions employed, only oxalic acid and EDTA were found to give any significant interference. The sensors were evaluated by carrying out citric acid determinations on spiked and unspiked samples of an acid citrate dextrose (ACD) formulation, lime flesh and juice. For lime juice, recoveries were calculated to be 96.8% (% CV = 2.7%) for a sample fortified with 5% citric acid and for ACD 99.4% (%CV = 2.6%) when fortified at 2.30% citric acid. Further studies showed the possibility of determining citric acid concentrations in lime juice and fruit directly, without the need for an added electrolyte. These performance characteristics indicate that reliable data may be obtained for citric acid measurements in such samples. To our knowledge, this is the first report on the electrocatalytic oxidation of citric acid and its application using a CoPC-SPCE.

Using Maize δ15N values to assess soil fertility in fifteenth- and sixteenth-century ad Iroquoian agricultural fields.

Native Americans developed agronomic practices throughout the Western Hemisphere adapted to regional climate, edaphic conditions, and the extent of dependence on agriculture for subsistence. These included the mounding or "corn hill" system in northeastern North America. Iroquoian language speakers of present-day New York, USA, and Ontario and Québec, Canada were among those who used this system. While well-known, there has been little archaeological documentation of the system. As a result, there is scant archaeological evidence on how Iroquoian farmers maintained soil fertility in their often-extensive agricultural fields. Using δ15N values obtained on fifteenth- and sixteenth-century AD maize kernels from archaeological sites in New York and Ontario, adjusted to take into account changes that result from charring as determined through experiments, we demonstrate that Iroquoian farmers were successful at maintaining nitrogen in their agricultural fields. These results add to our archaeological knowledge of Iroquoian agronomic practices. Our results also indicate the potential value of obtaining δ15N values on archaeological maize in the investigation of Native American agronomic practices.

Preparation of screen-printed electrochemical immunosensors for estradiol, and their application in biological fluids.

The method of fabrication of a prototype electrochemical immunosensor for estradiol (E2) is described. Methodologies are also given for colorimetric assays, which can be used to verify and optimize reagent performance, prior to their use in the electrochemical immunoassay: these include an E2 ELISA and a colorimetric assay performed on the immunosensor surface. The electrochemical immunosensor system uses screen-printed carbon electrodes (SPCEs) upon which antibody against E2 is immobilized. Antibodies (rabbit anti-mouse IgG, then monoclonal mouse anti-E2) are immobilized by passive adsorption onto the working electrode surface. A competitive immunoassay is then performed using an alkaline-phosphatase-labeled E2 conjugate. Electrochemical measurements are performed using differential pulse voltammetry (DPV) to detect the production of 1-naphthol from 1-naphthyl phosphate. The calibration plot of DPV peak current vs. E2 concentration shows a measurable range of 25-500 pg/mL with a detection limit of 50 pg/mL. The immunosensor can be applied to the determination of E2 in spiked serum, following an extraction step with diethyl ether.

Fish and maize: Bayesian mixing models of fourteenth- through seventeenth-century AD ancestral Wendat diets, Ontario, Canada.

Freshwater and marine fish have been important components of human diets for millennia. The Great Lakes of North America, their tributaries and smaller regional freshwater bodies are important Native American fisheries. The ethnohistorical record, zooarchaeological remains, and isotopic values on human bone and tooth collagen indicate the importance of fish in fourteenth- through seventeenth-century ancestral Wendat diets in southern Ontario, which is bordered by three of the Great Lakes. Maize (Zea mays ssp. mays) was the primary grain of Native American agricultural systems in the centuries prior to and following sustained European presence. Here we report new Bayesian dietary mixing models using previously published δ13C and δ15N values on ancestral Wendat bone and tooth collagen and tooth enamel. The results confirm previous estimates from δ13C values that ancestral Wendat diets included high proportions of maize but indicate much higher proportions of fish than has previously been recognized. The results also suggest that terrestrial animals contributed less to ancestral Wendat diets than is typically interpreted based on zooarchaeological records.

Radiocarbon, Bayesian chronological modeling and early European metal circulation in the sixteenth-century AD Mohawk River Valley, USA.

European metal artifacts in assemblages from sites predating the physical presence of Europeans in Northern Iroquoia in present-day New York, USA and southern Ontario, Canada have been used as chronological markers for the mid-sixteenth century AD. In the Mohawk River Valley of New York, European metal artifacts at sites pre-dating the physical presence of Europeans have been used by archaeologists as a terminus post quem (TPQ) of 1525 to 1550 in regional chronologies. This has been done under the assumption that these metals did not begin to circulate until after sustained European presence on the northern Atlantic coast beginning in 1517. Here we use Bayesian chronological modeling of a large set of radiocarbon dates to refine our understanding of early European metal circulation in the Mohawk River Valley. Our results indicate that European iron and cuprous metals arrived earlier than previously thought, by the beginning of the sixteenth century, and cannot be used as TPQs. Together with recent Bayesian chronological analyses of radiocarbon dates from several sites in southern Ontario, these results add to our evolving understanding of intra-regional variation in Northern Iroquoia of sixteenth-century AD circulation and adoption of European goods.

An analysis of network brokerage and geographic location in fifteenth-century AD Northern Iroquoia.

Iroquoian villagers living in present-day Jefferson County, New York, at the headwaters of the St. Lawrence River and the east shore of Lake Ontario, played important roles in regional interactions during the fifteenth century AD, as brokers linking populations on the north shore of Lake Ontario with populations in eastern New York. This study employs a social network analysis and least cost path analysis to assess the degree to which geographical location may have facilitated the brokerage positions of site clusters within pan-Iroquoian social networks. The results indicate that location was a significant factor in determining brokerage. In the sixteenth century AD, when Jefferson County was abandoned, measurable increases in social distance between other Iroquoian populations obtained. These results add to our understandings of the dynamic social landscape of fifteenth and sixteenth century AD northern Iroquoia, complementing recent analyses elsewhere of the roles played in regional interaction networks by populations located along geopolitical frontiers.