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

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

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

Do agronomic approaches aligned to regenerative agriculture improve the micronutrient concentrations of edible portions of crops? A scoping review of evidence

Regenerative Agriculture (RA) is used to describe nature-based agronomic approaches that aim to build soil health and crop resilience, minimize negative environmental outcomes, and improve farmer livelihoods. A benefit that is increasingly attributed to crops grown under RA practices is improved nutritional content. However, we do not know the extent to which RA influences crop nutritional quality and under what management approaches and context, can such effects be realized. A scoping review of recent literature (Web of Science, 2000-2021) was carried out to assess the evidence that RA approaches improve crop micronutrient quality. Papers included combinations of agronomic approaches that could be defined as Regenerative: "Organic Inputs" including composts and manures, cover crops, crop rotations, crop residues and biochars; "Reduced Tillage", "Intercropping", "Biostimulants" e.g. arbuscular mycorrhizal fungi; plant growth promoting bacteria, and "Irrigation", typically deficit-irrigation and alternate wetting and drying. The crop types reviewed were predetermined covering common sources of food and included: Tomato (Solanum lycopersicum L.), Wheat (Triticum aestivum L.), Rice (Oryza sativa L.), Maize (Zea mays L.), Pulses (Fabaceae), Alliums (Allium spp.), and "other" crop types (30 types). This scoping review supports a potential role for RA approaches in increasing the concentrations of micronutrients in the edible portions of several crop types under specific practices, although this was context specific. For example, rice grown under increased organic inputs showed significant increases in grain zinc (Zn) concentration in 15 out of 16 studies. The vitamin C concentration of tomato fruit increased in similar to 50% of studies when plants were grown under increased organic inputs, and in 76% of studies when plants were grown under deficit irrigation. Overall, the magnitude and reproducibility of the effects of RA practices on most crop nutritional profiles were difficult to assess due to the diversity of RA approaches, geographical conditions, and the limited number of studies for most crops in each of these categories. Future research with appropriate designs, improved on-farm surveillance and nutritional diagnostics are needed for better understanding the potential role of RA in improving the quality of food, human nutrition, and health

Unravelling the impact of soil types on zinc, iron, and selenium concentrations in grains and straw of wheat/Amblyopyrum muticum and wheat/Triticum urartu doubled haploid lines

The concentration of mineral nutrients in plants is associated with bioavailabilities of soil mineral nutrients, which are regulated by various soil physio-chemical properties. A pot experiment was conducted to investigate the effects of soil type on grain and straw zinc (Zn), iron (Fe) and selenium (Se) concentrations of wheat/Amblyopyrum muticum and wheat/Triticum urartu doubled haploid lines. A set of 42 treatments in a factorial combination with 21 genotypes and two soil types collected from Ngabu and Chitedze Research Stations in Malawi was laid in a randomised complete block design (RCBD) in three replicates. Pre-experiment soil Zn and Fe were extracted using DTPA extraction method followed by analysis with inductively coupled plasma-mass spectrometry (ICP-MS). Aqua-regia hotplate acid digestion was used to extract soil Se and analysis was done using ICPM-MS. Grain and straw samples were digested using nitric acid digestion (HNO3) and analysed using ICP-MS. Soil analysis results showed that the two soils had the same textural class (Sandy clay loam), but different mineral concentrations, pH levels and percentage organic matter. Analysis of variance revealed a ~two-fold higher Zn concentration in grains grown in low pH, high Zn soils (Chitedze soils) compared to grains grown in high pH, low Zn soils (Ngabu soils). Variation in grain Zn concentration was associated with the genotypes (p = 0002), soil type (p = <0.0001), and their interaction (p = 0.035). Grain Fe was 1.3-fold higher in low pH than in high pH soils, and it was influenced by genotypes (p = < 0.0001) and soil type (p = <0.0001). Grain Se was highly associated with soil type (p = <0.0001), and it was 30-fold higher in high pH than in low pH soils. Straw Zn was generally higher in plants grown in Chitedze soils than Ngabu soils, whilst straw Se was higher in plants grown in Ngabu soils than Chitedze soils. The findings demonstrate the significance of soil physio-chemical properties for mineral accumulation and distribution to plant parts, thus informing future breeding programs on importantconsiderations on crop genetic biofortification with the three mineral element

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

Novel sources of variation in grain Zinc (Zn) concentration in bread wheat germplasm derived from Watkins landraces

A diverse panel of 245 wheat genotypes, derived from crosses between landraces from the Watkins collection representing global diversity in the early 20th century and the modern wheat cultivar Paragon, was grown at two field sites in the UK in 2015–16 and the concentrations of zinc and iron determined in wholegrain using inductively coupled plasma-mass spectrometry (ICP-MS). Zinc concentrations in wholegrain varied from 24–49 mg kg-1 and were correlated with iron concentration (r = 0.64) and grain protein content (r = 0.14). However, the correlation with yield was low (r = -0.16) indicating little yield dilution. A sub-set of 24 wheat lines were selected from 245 wheat genotypes and characterised for Zn and Fe concentrations in wholegrain and white flour over two sites and years. White flours from 24 selected lines contained 8–15 mg kg-1 of zinc, which was positively correlated with the wholegrain Zn concentration (r = 0.79, averaged across sites and years). This demonstrates the potential to exploit the diversity in landraces to increase the concentration of Zn in wholegrain and flour of modern high yielding bread wheat cultivars

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

Clinical course, therapeutic responses and outcomes in relapsing MOG antibody-associated demyelination.

Abstract OBJECTIVE: We characterised the clinical course, treatment and outcomes in 59 patients with relapsing myelin oligodendrocyte glycoprotein (MOG) antibody-associated demyelination. METHODS: We evaluated clinical phenotypes, annualised relapse rates (ARR) prior and on immunotherapy and Expanded Disability Status Scale (EDSS), in 218 demyelinating episodes from 33 paediatric and 26 adult patients. RESULTS: The most common initial presentation in the cohort was optic neuritis (ON) in 54% (bilateral (BON) 32%, unilateral (UON) 22%), followed by acute disseminated encephalomyelitis (ADEM) (20%), which occurred exclusively in children. ON was the dominant phenotype (UON 35%, BON 19%) of all clinical episodes. 109/226 (48%) MRIs had no brain lesions. Patients were steroid responsive, but 70% of episodes treated with oral prednisone relapsed, particularly at doses <10\u2009mg daily or within 2 months of cessation. Immunotherapy, including maintenance prednisone (P=0.0004), intravenous immunoglobulin, rituximab and mycophenolate, all reduced median ARRs on-treatment. Treatment failure rates were lower in patients on maintenance steroids (5%) compared with non-steroidal maintenance immunotherapy (38%) (P=0.016). 58% of patients experienced residual disability (average follow-up 61 months, visual loss in 24%). Patients with ON were less likely to have sustained disability defined by a final EDSS of 652 (OR 0.15, P=0.032), while those who had any myelitis were more likely to have sustained residual deficits (OR 3.56, P=0.077). CONCLUSION: Relapsing MOG antibody-associated demyelination is strongly associated with ON across all age groups and ADEM in children. Patients are highly responsive to steroids, but vulnerable to relapse on steroid reduction and cessation