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Modifying the potato tuber storage protein patatin targeting improved thermal stability
Get PDFMain conclusionGene editing of the patatin gene cluster using a single-guide RNA sequence consistently modifies over 10% of the targeted genes in modified individuals.AbstractPatatins have gained recent attention, as a group of highly nutritious proteins with excellent functional properties. Some techniques have been suggested for industrial-scale patatin purification, mostly as a by-product from potato starch processing. The purification process has proved to be a challenge due to the low thermostability of patatins, especially under acidic conditions. One strategy to make patatin more accessible for extraction would be to stabilize the protein structure through the introduction of point mutations. Here, we show that the tuber expression of patatin genes is dominated by a few genes from the extended gene family, most of which were predicted to be catalytically inactive. We have further evaluated the suitability of the patatin gene cluster as a target for clustered regularly interspaced repeat (CRISPR)/Cas9-based mutagenesis. In the mutation study, we show that targeting using a single single-stranded guide RNA (sgRNA) can lead to mutations in over 10% of all alleles. Finally, four patatin variants with amino acid substitutions were designed based on in silico analysis of patatin protein structure. These modified patatins were then heterologously expressed in bacteria and evaluated for increased thermostability. While none of the mutant proteins performed better than a wild-type variant, with regard to their thermal properties, one candidate proved to be less sensitive to shifting pH, making it an interesting candidate for further optimizations
Reverse microdialysis of sucrose stimulates soil fungal and bacterial growth at the microscale
Get PDFBackgroundThe rhizosphere is a critical microenvironment that plays key roles in plant nutrient availability, largely due to root interactions with rhizospheric microbes. However, we lack suitable methods that can elucidate mechanisms determining rhizospheric community structure and function within the context of a dynamic, undisturbed soil. Microdialysis has been used for low intrusive soil nutrient sampling at the scale of a fine root, with small probes that also enable release of defined compounds. We evaluated whether microdialysis could simulate exudation, by the release of sucrose, and stimulate changes in a soil microbial community, allowing us to determine the microbes that responded most to carbon release.ResultsMicrodialysis successfully stimulated growth on probe surfaces of fungi and bacteria, which were extracted and sequenced for identification. Microbial growth was also visualized with scanning electron microscopy. The majority of the species stimulated were classified as fast growing or opportunistic, e.g. yeasts, moulds, proteobacteria and actinobacteriota, which are known to respond quickly (within days) to the release of simple sugars as exudates in the rhizosphere.ConclusionsThe study demonstrates the potential of using microdialysis as a tool to investigate interactions between root exudation and soil microbial community composition, initially for individual compounds and in the future for more complex compositions
Evaluating the automated measurement of abnormal rising and lying down behaviours in dairy cows using 3D pose estimation
Get PDFThe structure of cubicles can hinder cows’ movements when transitioning between postures, leading to atypical motion patterns. Assessing posture transitions relies on visual observations. This study presents a framework for complementing these assessments with kinematic measurements using 3D pose estimation. A total 809 rising and 791 lying down posture transitions were recorded over 12 cubicles by 7 synchronized cameras and processed with 3D pose estimation locating the position of the poll, withers, T13 and sacrum. First, the displacement of the keypoints was used to detect phases of the posture transitions. This detection was compared with visual observations of 200 recordings. The average mean absolute difference in detected timestamps between human and machine across all phases was 0.5 s (average σ = 0.7) and was under 0.9 s for all phases. Second, indicators were scored based on spatial use and duration, and their distribution compared to existing thresholds. We observed that 59.9 % of rising bouts and 29.1 % of lying down bouts exceeded at least one threshold. Rising delay occurred in 2.8 % of rising bouts and backwards crawling in 59.2 %. Lying down duration exceeded the threshold in 28.9 % of bouts, and rear limbs shifting duration in 8.3 %. Side lunge had a binary threshold which was not adapted to continuous sensor data. Finally, we investigated the association between indicators and found distinct dimensions for head lunge and crawling. We conclude that 3D pose is useful to score posture transition indicators, and that several indicators should be used together to capture distinct dimensions
Coupling Between Methylmercury and Carbon-Gases Across Boreal Rivers of Québec, Canada
Get PDFWe examine correlations between mercury (Hg) and carbon (C) concentrations and speciation by synthesizing 526 river water samples across three distinct regions in Qu & eacute;bec, Canada (48 degrees to 55 degrees N and 62 degrees to 80 degrees W). Positive correlations between methylmercury (MeHg) per unit Hg and C-gases per unit dissolved organic carbon suggest Hg-C co-transformation within rivers. Correlations between MeHg and methane (CH4) concentrations strengthen when adjusted for oxidation, revealing an association that goes beyond the established link between fermentation and Hg methylation in anoxic environments. These correlations persist but are manifested differently across regions. Colder, wetland-dominated regions indicated stronger terrestrial co-loading, while warmer and more recently impounded systems exhibited more co-transformation. These findings emphasize the interconnected biogeochemical cycles of Hg-C in northern rivers and highlight the importance of landscape characteristics influencing their coupling
Elevated CO2 decreases micronutrient Zn but not Fe in vegetables - evidence from a meta-analysis
Get PDFWith carbon dioxide (CO2) levels continuing to rise in the coming decades and threatening agro-ecosystems worldwide, it is crucial to understand the impact of elevated CO2 on global food production and security. Elevated CO2 levels have been found to reduce micronutrients such as Zinc (Zn) and Iron (Fe) in staple crops, potentially exacerbating the already existing global micronutrient deficiency issue. However, as vegetables serve as another key source of micronutrients, it remains uncertain to what extent this negative effect on micronutrient levels also applies to them. To address this, we investigated the effects of elevated CO2 on Zn and Fe in vegetables using a meta-analysis. As expected, we found a significant increase (27%, 95% CI: 14-41%) in vegetable biomass production under elevated CO2 levels. Elevated CO2 (i) significantly reduced overall Zn concentration in vegetables by 8.9% (95% CI: 4-14%), while this effect was pronounced only in fruit vegetables (11%), but not in leafy and stem vegetables; (ii) consistently exhibited minimal effects on Fe concentration in vegetables. In the context of climate change with rising CO2 levels, these findings suggest that elevated CO2 could potentially exacerbate Zn deficiencies through vegetable consumption, albeit with enhanced vegetable yields. Furthermore, as the global population increasingly adopts vegetarian diets in the future, these results underscore the need for mitigation strategies to address potential future micronutrient deficiencies
Quantification and comparison of subnational and national agricultural nitrogen flows in Denmark and Sweden
Get PDFEnsuring food production with low nitrogen (N) environmental emissions requires good quantitative knowledge of N flows in agricultural systems to monitor emissions and N use efficiency (NUE, the ratio of N outputs to inputs). Our study quantifies the main N agricultural flows at subnational and national scales in Denmark and Sweden from 2011 to 2020, calculating the NUE for crop and livestock production and associated nitrous oxide (N2O) and ammonia (NH3) emissions. In Denmark, our results indicate a similar use of organic (manure) and synthetic N fertilizers (230 and 213 kt N y-1, 83 and 77 kg N ha-1 y-1); in contrast, Sweden used more synthetic (162 kt N y-1, 54 kg N ha-1 y-1) than organic N (108 kt N y-1, 36 kg N ha-1 y-1), with subnational variation in manure use as determined by livestock population. Livestock feed N intake was twice as large in Denmark (384 kt N y-1) as in Sweden (176 kt N y-1), reflecting Denmark's larger livestock population. Denmark's national crop NUE was lower (0.51) than Sweden's (0.72), likely due to a lower proportion of grass-clover leys, higher N input rates, and more intensive production systems. However, considerable subnational variation existed in both countries. The livestock NUE was 0.29 in Denmark and 0.25 in Sweden; these differences are mainly due to a higher proportion of ruminants in Sweden with lower N feed use efficiency than pigs. Sweden emitted less N2O and NH3 per unit area (similar to 56% for both gases) and in total (similar to 52% for both gases) than Denmark due to lower use of N inputs and less intensive farming systems. West Denmark and South Sweden were identified as emission hotspots. Our research provides essential information at subnational and national scales to improve N management and reduce gaseous N pollution, supporting the transition towards more sustainable agroecosystems in Denmark and Sweden
Influence of TEMPO on preparation of softwood nanofibrils and their hydrogel network properties
Get PDFFrom an economic and environmental perspective, the use of less chemicals in the production of cellulose nanofibrils (CNFs) is advantageous. In this study, we investigated the oxidation (TEMPO/NaClO2/NaClO, pH 6.8) of softwood (SW) particles with varying amounts of TEMPO (16, 8 or 0 mg g(-1) of wood). Following, TEMPO-oxidized SW nanofibrils (TO-SWNFs) were obtained by nanofibrillation and their size, morphology, and crystallite size were assessed. Hydrogel networks of TO-SWNFs were prepared and mechanical properties were measured in dH(2)O and phosphate buffered saline (PBS) to compare their performance for possible biomedical applications such as wound dressings. The results reveal that the presence of TEMPO is of importance for TO-SWNF network properties, presenting higher eq. H2O absorption (approximate to 2500 %) and elongation at break (approximate to 10 %) with good wet strength (approximate to 180 kPa). In addition, a decrease in use of TEMPO catalyst from 16 to 8 mg g(-1) of wood is possible, without detrimental effects on hydrogel network properties (dH(2)O absorption approximate to 2000 %, elongation at break approximate to 13 %, wet strength approximate to 190 kPa) related to applications as wound dressings
Bioraffinering av ensilage
Get PDFBeroendet av importerad soja och behovet av mer varierande växtföljder i slättlandskapet har gjort att intresset för bioraffinering ökat. Om ensilage bioraffineras skulle bioraffineringsanläggningen kunna köras året om och det skulle förbättra ekonomin för en sådan anläggning. Syftet med denna studie var att kartlägga nivåer och variationen i näring med fokus på protein och enskilda aminosyror primärt i pressjuice.Ensilage från fyra olika silos bioraffinerades och utbytesberäkningar gjordes. Aminosyra innehållet i ensilage, presskaka och pressjuice analyserades. Av råproteinet hamnade 10–35 % i pressjuicen och 17–50 % av askan. Ensilagets torrsubstanshalt hade stor betydelse för utbytet. Torrt ensilage gav lågt utbyte. Innehållet av den för grisar viktiga aminosyran lysin i pressjuicen var mellan 4,9 och 6,2 % av råproteinet. Halterna av de för fjäderfä viktiga aminosyrorna cystein och metionin var låga eller ej mätbara. Innehållet av övriga aminosyror redovisas i rapporten. Variationen i proteinkvalitet mellan de olika silorna och resultat från andra studier var stor. Analys av aminosyror och annat innehåll i pressjuicen är nödvändigt för att kunna erbjuda djuren balanserade foderstater
Circular causality in volition
Get PDFConventional scientific paradigms predominantly emphasize upward causality, often overlooking or dismissing the role of downward causality. This approach is also prevalent in neuroscience, where cortical neurodynamics and higher cognitive functions are typically viewed as consequences of neuronal or even ion channel activity. Conversely, mental phenomena are generally assumed to lack causal efficacy over neural processes-an assumption that is increasingly being questioned. The causality associated with volition may be analyzed at three organizational levels: (1) neuronal interactions within cortical networks, (2) interregional dynamics between distinct brain areas, and (3) the reciprocal relationship between the nervous system and its environmental context. Across all these domains, circular rather than strictly linear causality appears to be at play. This paper examines the implications of such circular causality for volition and the longstanding problem of free will, with particular reference to insights derived from neurocomputational modeling