Optimising the analysis of transcript data using high density oligonucleotide arrays and genomic DNA-based probe selection

Background: Affymetrix GeneChip arrays are widely used for transcriptomic studies in a diverse range of species. Each gene is represented on a GeneChip array by a probe-set, consisting of up to 16 probe-pairs. Signal intensities across probe-pairs within a probe-set vary in part due to different physical hybridisation characteristics of individual probes with their target labelled transcripts. We have previously developed a technique to study the transcriptomes of heterologous species based on hybridising genomic DNA (gDNA) to a GeneChip array designed for a different species, and subsequently using only those probes with good homology. Results: Here we have investigated the effects of hybridising homologous species gDNA to study the transcriptomes of species for which the arrays have been designed. Genomic DNA from Arabidopsis thaliana and rice (Oryza sativa) were hybridised to the Affymetrix Arabidopsis ATH1 and Rice Genome GeneChip arrays respectively. Probe selection based on gDNA hybridisation intensity increased the number of genes identified as significantly differentially expressed in two published studies of Arabidopsis development, and optimised the analysis of technical replicates obtained from pooled samples of RNA from rice. Conclusion: This mixed physical and bioinformatics approach can be used to optimise estimates of gene expression when using GeneChip arrays

Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species

High-density oligonucleotide (oligo) arrays are a powerful tool for transcript profiling. Arrays based on GeneChip® technology are amongst the most widely used, although GeneChip® arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip® array is available, using a GeneChip® array designed for Arabidopsis thaliana (L.) Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip® array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM) probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P) stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip® array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affymetrix.arabidopsis.info/xspecies/ webcite and may be used to facilitate transcriptomic analyses of a wide range of plant and animal species in the absence of custom arrays

A spatial analysis of lime resources and their potential for improving soil magnesium concentrations and pH in grassland areas of England and Wales

Magnesium (Mg) is essential for animal health. Low Mg status (hypomagnesaemia) can be potentially fatal in ruminants, like cattle and sheep, and is widespread in Europe with economic impacts on farming. The application of Mg-rich agricultural lime products can help to ensure pasture forage consumed by animals contains sufficient Mg and, in areas of low pH, has the dual benefit of reducing soil acidity to levels best suited for grass production. This aim of this study was to determine if Mg-rich lime products could be used in a more effective manner in agricultural production systems. Potential resources of carbonate rocks (limestone, dolostone and chalk) in the UK, and their Mg:Ca status were identified, using datasets from the British Geological Survey (BGS). These data were combined with the locations of agricultural lime quarries, and areas where soils are likely to be deficient in Mg and/or require liming. Areas of potential demand for Mg-rich agricultural lime include areas in south east Wales, the Midlands and North East England. Although, areas where this may be an effective solution to low soil Mg values are restricted by the availability of suitable products. Conversely, areas of low soil pH in England and Wales are often found close to quarries with the ability to supply high Ca limes, suggesting that the low rates of lime use and liming is not due to supply factors. This study provides information that can help to guide on-farm decision making for use of Mg-rich and other lime resources. This could be used in conjunction with other options to reduce risks of Mg deficiency in livestock, and improve soil pH

Evidence of neutral transcriptome evolution in plants

The transcriptome of an organism is its set of gene transcripts (mRNAs) at a defined spatial and temporal locus. Because gene expression is affected markedly by environmental and developmental perturbations, it is widely assumed that transcriptome divergence among taxa represents adaptive phenotypic selection. This assumption has been challenged by neutral theories which propose that stochastic processes drive transcriptome evolution. To test for evidence of neutral transcriptome evolution in plants, we quantified 18 494 gene transcripts in nonsenescent leaves of 14 taxa of Brassicaceae using robust cross-species transcriptomics which includes a two-step physical and in silicobased normalization procedure based on DNA similarity among taxa. Transcriptome divergence correlates positively with evolutionary distance between taxa and with variation in gene expression among samples. Results are similar for pseudogenes and chloroplast genes evolving at different rates. Remarkably, variation in transcript abundance among root-cell samples correlates positively with transcriptome divergence among root tissues and among taxa. Because neutral processes affect transcriptome evolution in plants, many differences in gene expression among or within taxa may be nonfunctional, reflecting ancestral plasticity and founder effects. Appropriate null models are required when comparing transcriptomes in space and time

Differences in the nutritional quality of improved finger millet genotypes in Ethiopia

Improved crop genotypes are constantly introduced. However, information on their nutritional quality is generally limited. The present study reports the proximate composition and the concentration and relative bioavailability of minerals of improved finger millets of different genotypes. Grains of finger millet genotypes (n = 15) grown in research station during 2019 and 2020 in Ethiopia, and replicated three times in a randomized complete block design, were analysed for proximate composition, mineral concentration (iron, zinc, calcium, selenium), and antinutritional factors (phytate, tannin and oxalate). Moreover, the antinutritional factors to mineral molar ratio method was used to estimate mineral bioavailability. The result shows a significant genotypic variation in protein, fat and fibre level, ranging from 10% to 14.6%, 1.0 to 3.8%, and 1.4 to 4.6%, respectively. Similarly, different finger millets genotypes had significantly different mineral concentrations ranging from 3762 ± 332 to 5893 ± 353 mg kg−1 for Ca, 19.9 ± 1.6 to 26.2 ± 2.7 mg kg−1 for Zn, 36.3 ± 4.6 to 52.9 ± 9.1 mg kg−1 for Fe and 36.6 ± 11 to 60.9 ± 22 µg kg−1 for Se. Phytate (308–360 µg g−1), tannin (0.15–0.51 mg g−1) and oxalate (1.26–4.41 mg g−1) concentrations were also influenced by genotype. Antinutritional factors to minerals molar ratio were also significantly different by genotypes but were below the threshold for low mineral bioavailability. Genotype significantly influenced mineral and antinutritional concentrations of finger millet grains. In addition, all finger millet genotypes possess good mineral bioavailability. Especially, the high Ca concentration in finger millet, compared to in other cereals, could play a vital role to combating Ca deficiency. The result suggests the different finger millet genotypes possess good nutrient content and may contribute to the nutrition security of the local people

Linkages between soil, crop, livestock, and human selenium status in Sub-Saharan Africa: a scoping review.

Selenium (Se) is essential for human health, however, data on population Se status and agriculture-nutrition-health linkages are limited in sub-Saharan Africa (SSA). The scoping review aims to identify linkages between Se in soils/crops, dietary Se intakes, and livestock and human Se status in SSA. Online databases, organisational websites and grey literature were used to identify articles. Articles were screened at title, abstract and full text levels using eligibility criteria. The search yielded 166 articles from which 112 were excluded during abstract screening and 54 full text articles were assessed for eligibility. The scoping review included 34 primary studies published between 1984 and 2021. The studies covered Se concentrations in soils (n = 7), crops (n = 9), animal tissues (n = 2), livestock (n = 3), and human Se status (n = 15). The evidence showed that soil/crop Se concentrations affected Se concentration in dietary sources, dietary Se intake and biomarkers of Se status. Soil types are a primary driver of human Se status and crop Se concentration correlates positively with biomarkers of Se dietary status. Although data sets of Se concentrations exist across the food system in SSA, there is limited evidence on linkages across the agriculture-nutrition nexus. Extensive research on Se linkages across the food chain is warranted

Immersion mode heterogeneous ice nucleation by an illite rich powder representative of atmospheric mineral dust

Atmospheric dust rich in illite is transported globally from arid regions and impacts cloud properties through the nucleation of ice. We present measurements of ice nucleation in water droplets containing known quantities of an illite rich powder under atmospherically relevant conditions. The illite rich powder used here, NX illite, has a similar mineralogical composition to atmospheric mineral dust sampled in remote locations, i.e. dust which has been subject to long range transport, cloud processing and sedimentation. Arizona Test Dust, which is used in other ice nucleation studies as a model atmospheric dust, has a significantly different mineralogical composition and we suggest that NX illite is a better surrogate of natural atmospheric dust. Using optical microscopy, heterogeneous nucleation in the immersion mode by NX illite was observed to occur dominantly between 246 K and the homogeneous freezing limit. In general, higher freezing temperatures were observed when larger surface areas of NX illite were present within the drops. Homogenous nucleation was observed to occur in droplets containing low surface areas of NX illite. We show that NX illite exhibits strong particle to particle variability in terms of ice nucleating ability, with ~1 in 105 particles dominating ice nucleation when high surface areas were present. In fact, this work suggests that the bulk of atmospheric mineral dust particles may be less efficient at nucleating ice than assumed in current model parameterisations. For droplets containing ≤2 × 10−6 cm2 of NX illite, freezing temperatures did not noticeably change when the cooling rate was varied by an order of magnitude. The data obtained during cooling experiments (surface area ≤2 × 10−6 cm2) is shown to be inconsistent with the single component stochastic model, but is well described by the singular model (ns(236.2 K ≤ T ≤ 247.5 K) = exp(6.53043 × 104− 8.2153088 × 102T + 3.446885376T2 − 4.822268 × 10−3T3). However, droplets continued to freeze when the temperature was held constant, which is inconsistent with the time independent singular model. We show that this apparent discrepancy can be resolved using a multiple component stochastic model in which it is assumed that there are many types of nucleation sites, each with a unique temperature dependent nucleation coefficient. Cooling rate independence can be achieved with this time dependent model if the nucleation rate coefficients increase very rapidly with decreasing temperature, thus reconciling our measurement of nucleation at constant temperature with the cooling rate independence

Plant Available Zinc Is Influenced by Landscape Position in the Amhara Region, Ethiopia

Zinc (Zn) is an important element determining the grain quality of staple food crops and deficient in many Ethiopian soils. However, farming systems are highly variable in Ethiopia due to different soil types and landscape cropping positions. Zinc availability and uptake by plants from soil and fertilizer sources are governed by the retention and release potential of the soil, usually termed as adsorption and desorption, respectively. The aim of this study was to characterize the amount of plant available Zn at different landscape positions. During the 2018/19 cropping season, adsorption-desorption studies were carried out on soil samples collected from on-farm trials conducted at Aba Gerima, Debre Mewi and Markuma in the Amhara Region. In all locations and landscape positions, adsorption and desorption increased with increasing Zn additions. The amount of adsorption and desorption was highly associated with the soil pH, the soil organic carbon concentration and cation exchange capacity, and these factors are linked to landscape positions. The Freundlich isotherm fitted very well to Zn adsorption (r2 0.87–0.99) and desorption (r2 0.92–0.99), while the Langmuir isotherm only fitted to Zn desorption (r2 0.70–0.93). Multiple regression models developed by determining the most influential soil parameters for Zn availability could be used to inform Zn fertilizer management strategies for different locations and landscape positions in this region, and thereby improve plant Zn use efficienc

Diet and feeding in the Cape Cobra, Naja nivea

It is widely reported that African cobras (genus Naja) are generalist predators, but examination of formal literature yields relatively little evidence of this for most species. Here, we review the diet of cape cobras (Naja nivea) based on examination of museum specimens, an extensive literature review, and social media reported natural history observations. We quantify dietary niche breadth and test the hypothesis that cape cobras are generalist predators. Additionally, we test the hypotheses that diet varies significantly (1) across biomes, and (2) across winter, summer and year-round rainfall regions. We gathered 101 feeding records for cape cobras and based on family-level prey frequencies calculated Levins’ measure of niche breadth as B = 6.57, which we standardised to BA = 0.29. We found no association between prey classes and biomes or rainfall region. Our data indicate that cape cobras consume a wide range of vertebrate prey from all four tetrapod classes, including a large number and diversity of snakes. Our findings suggest that cape cobras may be involved in several important ecological processes. Moreover, our study demonstrates the utility and value of collating natural history observations reported on social media platforms

Route of administration affects corticosteroid sensitivity of a combined ovalbumin and lipopolysaccharide model of asthma exacerbation in guinea-pigs

Lipopolysaccharide (LPS) contributes to asthma exacerbations and development of inhaled corticosteroid insensitivity. Complete resistance to systemic corticosteroids is rare and most patients lie on a continuum of steroid responsiveness. The objective of this study was to examine the sensitivity of combined ovalbumin- (Ova) and LPS-induced functional and inflammatory responses to inhaled and systemic corticosteroid in conscious guinea-pigs, to test the hypothesis that the route of administration affects its sensitivity. Guinea-pigs were sensitised to Ova and challenged with inhaled Ova alone or combined with LPS. Airways function was determined by measuring specific airways conductance via whole-body plethysmography. Airways hyperresponsiveness to histamine was determined pre- and 24h post-Ova challenge. Airways inflammation and underlying mechanisms were determined from bronchoalveolar lavage cell counts and lung tissue cytokines. Vehicle or dexamethasone was administered by once-daily intraperitoneal injection (5, 10 or 20 mg/kg) or twice-daily inhalation (4 or 20 mg/ml) for 6 days before Ova challenge or Ova with LPS. LPS exacerbated Ova-induced responses, elongating early asthmatic responses (EAR), prolonging bronchoconstriction by histamine and further elevating airways inflammation. Intraperitoneal dexamethasone (20 mg/kg) significantly reduced the elongated EAR and airways inflammation but not the increased bronchoconstriction to histamine. In contrast, inhaled dexamethasone (20 mg/ml), which inhibited responses to Ova alone, did not significantly reduce functional and inflammatory responses to combined Ova and LPS. Combined Ova and LPS-induced functional and inflammatory responses are insensitive to inhaled but only partially sensitive to systemic dexamethasone. These results suggest that the route of corticosteroid administration may be important in determining the sensitivity of asthmatic responses to these agents