Chitosan from Mushroom Improves Drought Stress Tolerance in Tomatoes.

Chitosan is a derivative of chitin that is one of the most abundant biopolymers in nature, found in crustacean shells as well as in fungi cell walls. Most of the commercially available chitosans are produced from the exoskeletons of crustaceans. The extraction process involves harsh chemicals, has limited potential due to the seasonal and limited supply and could cause allergic reactions. However, chitosan has been shown to alleviate the negative effect of environmental stressors in plants, but there is sparse evidence of how chitosan source affects this bioactivity. The aim of this study was to investigate the ability of chitosan from mushroom in comparison to crustacean chitosan in enhancing drought stress tolerance in tomato plants (cv. MicroTom). Chitosan treatment was applied through foliar application and plants were exposed to two 14-day drought stress periods at vegetative and fruit set growth stages. Phenotypic (e.g., fruit number and weight), physiological (RWC) and biochemical-stress-related markers (osmolytes, photosynthetic pigments and malondialdehyde) were analyzed at different time points during the crop growth cycle. Our hypothesis was that this drought stress model will negatively impact tomato plants while the foliar application of chitosan extracted from either crustacean or mushroom will alleviate this effect. Our findings indicate that drought stress markedly decreased the leaf relative water content (RWC) and chlorophyll content, increased lipid peroxidation, and significantly reduced the average fruit number. Chitosan application, regardless of the source, improved these parameters and enhanced plant tolerance to drought stress. It provides a comparative study of the biostimulant activity of chitosan from diverse sources and suggests that chitosan sourced from fungi could serve as a more sustainable and environmentally friendly alternative to the current chitosan from crustaceans.

The bioaccessibility and tolerability of marine-derived sources of magnesium and calcium.

It is generally accepted that mineral deficiencies, including magnesium and calcium, are widespread globally. Dietary supplementation may be an effective approach to combat such deficiencies. However, challenges associated with limited mineral solubility in the digestive system can impede effective dissolution and hinder absorption, leading to deficiency, and undesirable gastrointestinal disturbances including diarrhoea. Seawater is considered to be a rich source of bioactive magnesium, calcium, and 72 other trace minerals. In this study, we examine two different marine-derived multimineral products as potential dietary supplements. Aquamin-Mg, sourced from seawater is rich in magnesium (12%), and Aquamin F, a seaweed-derived multimineral is rich in calcium (32%). Both products also contain a diverse array of over 72 minerals, characteristic of their oceanic origin. Our study comprises two experiments. The first experiment evaluates and compares the solubility of Aquamin-Mg, commercially available magnesium bisglycinate, and Pure Magnesium Bisglycinate (PrizMAG) during in vitro digestion using the INFOGEST method. Results demonstrate that Aquamin-Mg exhibits superior solubility than the other magnesium sources during the gastric and intestinal phases, particularly when administered alongside food materials. The second experiment is a randomized, double-blind, placebo-controlled study in a small cohort of healthy older aged adults to assess the tolerability of a combined Aquamin-Mg/Aquamin-F supplement over a 12-week period. The findings indicate that this combination supplement is well-tolerated, with no significant adverse events reported, emphasizing its potential as a means of addressing mineral deficiencies.

A Review of Potential Feed Additives Intended for Carbon Footprint Reduction through Methane Abatement in Dairy Cattle.

Eight rumen additives were chosen for an enteric methane-mitigating comparison study including garlic oil (GO), nitrate, Ascophyllum nodosum (AN), Asparagopsis (ASP), Lactobacillus plantarum (LAB), chitosan (CHI), essential oils (EOs) and 3-nitrooxypropanol (3-NOP). Dose-dependent analysis was carried out on selected feed additives using a meta-analysis approach to determine effectiveness in live subjects or potential efficacy in live animal trials with particular attention given to enteric gas, volatile fatty acid concentrations, and rumen microbial counts. All meta-analysis involving additives GO, nitrates, LAB, CHI, EOs, and 3-NOP revealed a reduction in methane production, while individual studies for AN and ASP displayed ruminal bacterial community improvement and a reduction in enteric CH4. Rumen protozoal depression was observed with GO and AN supplementation as well as an increase in propionate production with GO, LAB, ASP, CHI, and 3-NOP rumen fluid inoculation. GO, AN, ASP, and LAB demonstrated mechanisms in vitro as feed additives to improve rumen function and act as enteric methane mitigators. Enzyme inhibitor 3-NOP displays the greatest in vivo CH4 mitigating capabilities compared to essential oil commercial products. Furthermore, this meta-analysis study revealed that in vitro studies in general displayed a greater level of methane mitigation with these compounds than was seen in vivo, emphasising the importance of in vivo trials for final verification of use. While in vitro gas production systems predict in vivo methane production and fermentation trends with reasonable accuracy, it is necessary to confirm feed additive rumen influence in vivo before practical application.

Predictive modelling of brain disorders with magnetic resonance imaging: A systematic review of modelling practices, transparency, and interpretability in the use of convolutional neural networks.

Brain disorders comprise several psychiatric and neurological disorders which can be characterized by impaired cognition, mood alteration, psychosis, depressive episodes, and neurodegeneration. Clinical diagnoses primarily rely on a combination of life history information and questionnaires, with a distinct lack of discriminative biomarkers in use for psychiatric disorders. Symptoms across brain conditions are associated with functional alterations of cognitive and emotional processes, which can correlate with anatomical variation; structural magnetic resonance imaging (MRI) data of the brain are therefore an important focus of research, particularly for predictive modelling. With the advent of large MRI data consortia (such as the Alzheimer's Disease Neuroimaging Initiative) facilitating a greater number of MRI-based classification studies, convolutional neural networks (CNNs)-deep learning models well suited to image processing tasks-have become increasingly popular for research into brain conditions. This has resulted in a myriad of studies reporting impressive predictive performances, demonstrating the potential clinical value of deep learning systems. However, methodologies can vary widely across studies, making them difficult to compare and/or reproduce, potentially limiting their clinical application. Here, we conduct a qualitative systematic literature review of 55 studies carrying out CNN-based predictive modelling of brain disorders using MRI data and evaluate them based on three principles-modelling practices, transparency, and interpretability. We propose several recommendations to enhance the potential for the integration of CNNs into clinical care.

Effects of an Extract of the Brown Seaweed Ascophylum nodosum on Postprandial Glycaemic Control in Healthy Subjects: A Randomized Controlled Study.

The effects of the consumption of an extract of the brown seaweed Ascophyllum nodosum (BSW) on postprandial glucose and insulin responses to white bread were investigated in an acute, randomized, double-blind, three-arm, crossover, controlled trial in healthy, normoglycemic subjects. Sixteen subjects were administered either control white bread (50 g total digestible carbohydrates) or white bread with 500 mg or 1000 mg of BSW extract. Biochemical parameters were measured in venous blood over 3 h. Significant inter-individual variation in the glycaemic response to white bread was observed. Analysis of the responses of all subjects to either 500 mg or 1000 mg of BSW extract versus control revealed no significant effects of treatments. The variation in response to the control was used to classify individuals into glycaemic responders and non-responders. In the sub-cohort of 10 subjects with peak glucose levels after white bread above 1 mmol/L, we observed a significant decrease in maximum levels of plasma glucose after the intervention meal with 1000 mg of extract compared with the control. No adverse effects were reported. Further work is warranted to define all factors that determine "responders" to the effects of brown seaweed extracts and identify the cohort that would benefit the most from their consumption.

Transcriptome, Biochemical and Phenotypic Analysis of the Effects of a Precision Engineered Biostimulant for Inducing Salinity Stress Tolerance in Tomato.

Salinity stress is a major problem affecting plant growth and crop productivity. While plant biostimulants have been reported to be an effective solution to tackle salinity stress in different crops, the key genes and metabolic pathways involved in these tolerance processes remain unclear. This study focused on integrating phenotypic, physiological, biochemical and transcriptome data obtained from different tissues of Solanum lycopersicum L. plants (cv. Micro-Tom) subjected to a saline irrigation water program for 61 days (EC: 5.8 dS/m) and treated with a combination of protein hydrolysate and Ascophyllum nodosum-derived biostimulant, namely PSI-475. The biostimulant application was associated with the maintenance of higher K+/Na+ ratios in both young leaf and root tissue and the overexpression of transporter genes related to ion homeostasis (e.g., NHX4, HKT1;2). A more efficient osmotic adjustment was characterized by a significant increase in relative water content (RWC), which most likely was associated with osmolyte accumulation and upregulation of genes related to aquaporins (e.g., PIP2.1, TIP2.1). A higher content of photosynthetic pigments (+19.8% to +27.5%), increased expression of genes involved in photosynthetic efficiency and chlorophyll biosynthesis (e.g., LHC, PORC) and enhanced primary carbon and nitrogen metabolic mechanisms were observed, leading to a higher fruit yield and fruit number (47.5% and 32.5%, respectively). Overall, it can be concluded that the precision engineered PSI-475 biostimulant can provide long-term protective effects on salinity stressed tomato plants through a well-defined mode of action in different plant tissues.

Potential Biomarkers of Acute Ischemic Stroke Etiology Revealed by Mass Spectrometry-Based Proteomic Characterization of Formalin-Fixed Paraffin-Embedded Blood Clots.

Background and Aims: Besides the crucial role in the treatment of acute ischemic stroke (AIS), mechanical thrombectomy represents a unique opportunity for researchers to study the retrieved clots, with the possibility of unveiling biological patterns linked to stroke pathophysiology and etiology. We aimed to develop a shotgun proteomic approach to study and compare the proteome of formalin-fixed paraffin-embedded (FFPE) cardioembolic and large artery atherosclerotic (LAA) clots. Methods: We used 16 cardioembolic and 15 LAA FFPE thrombi from 31 AIS patients. The thrombus proteome was analyzed by label-free quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant v1.5.2.8 and Perseus v.1.6.15.0 were used for bioinformatics analysis. Protein classes were identified using the PANTHER database and the STRING database was used to predict protein interactions. Results: We identified 1,581 protein groups as part of the AIS thrombus proteome. Fourteen significantly differentially abundant proteins across the two etiologies were identified. Four proteins involved in the ubiquitin-proteasome pathway, blood coagulation or plasminogen activating cascade were identified as significantly abundant in LAA clots. Ten proteins involved in the ubiquitin proteasome-pathway, cytoskeletal remodeling of platelets, platelet adhesion or blood coagulation were identified as significantly abundant in cardioembolic clots. Conclusion: Our results outlined a set of 14 proteins for a proof-of-principle characterization of cardioembolic and LAA FFPE clots, advancing the proteome profile of AIS human thrombi and understanding the pathophysiology of ischemic stroke.

Investigation of the direct effect of a precision Ascophyllum nodosum biostimulant on nitrogen use efficiency in wheat seedlings.

Reduction in the greenhouse gas (GHG) emissions and nitrogen (N) pollution of ground water by improving nitrogen use efficiency (NUE) in crops has become an intensively investigated research topic in pursuit of a more sustainable future. Although, distinct solutions have been proposed there are only a few reports documenting the detailed interplay between observed plant growth dynamics and changes in plant N related transcriptional and biochemical changes. It was previously demonstrated that the application of a formulated biostimulant (PSI-362) derived from Ascophyllum nodosum (ANE) improves N uptake in Arabidopsis thaliana and in barley. In this study, the effect of PSI-362 on the growth dynamics of wheat seedlings was evaluated at different biostimulant and N supplementation rates. Wheat grown on N deficient MS medium was also analysed from the first hour of the treatment until the depletion of the nutrients in the medium 9 days later. During this time the biomass increase measured for PSI-362 treated plants versus untreated controls was associated with increased nitrate uptake, with surplus N assimilated by the biomass in the form of glutamate, glutamine, free amino acids, soluble proteins, and chlorophyll. Phenotypical and biochemical analysis were supported by evaluation of differential expression of genetic markers involved in nitrate perception and transport (TaNRT1.1/NPF6.3), nitrate and nitrite reduction (TaNR1 and TaNiR1) and assimilation (TaGDH2, TaGoGAT, TaGS1). Finally, a comparative analysis of the precision biostimulant PSI-362 and two generic ANEs demonstrated that the NUE effect greatly differs depending on the ANE formulation used.

Deep Learning of Histopathological Features for the Prediction of Tumour Molecular Genetics.

Advanced diagnostics are enabling cancer treatments to become increasingly tailored to the individual through developments in immunotherapies and targeted therapies. However, long turnaround times and high costs of molecular testing hinder the widespread implementation of targeted cancer treatments. Meanwhile, gold-standard histopathological assessment carried out by a trained pathologist is widely regarded as routine and mandatory in most cancers. Recently, methods have been developed to mine hidden information from histopathological slides using deep learning applied to scanned and digitized slides; deep learning comprises a collection of computational methods which learn patterns in data in order to make predictions. Such methods have been reported to be successful in a variety of cancers for predicting the presence of biomarkers such as driver mutations, tumour mutational burden, and microsatellite instability. This information could prove valuable to pathologists and oncologists in clinical decision making for cancer treatment and triage for in-depth sequencing. In addition to identifying molecular features, deep learning has been applied to predict prognosis and treatment response in certain cancers. Despite reported successes, many challenges remain before the clinical implementation of such diagnostic strategies in the clinical setting is possible. This review aims to outline recent developments in the field of deep learning for predicting molecular genetics from histopathological slides, as well as to highlight limitations and pitfalls of working with histopathology slides in deep learning.

Reducing Nitrogen Input in Barley Crops While Maintaining Yields Using an Engineered Biostimulant Derived From Ascophyllum nodosum to Enhance Nitrogen Use Efficiency.

Intensive agricultural production utilizes large amounts of nitrogen (N) mineral fertilizers that are applied to the soil to secure high crop yields. Unfortunately, up to 65% of this N fertilizer is not taken up by crops and is lost to the environment. To compensate these issues, growers usually apply more fertilizer than crops actually need, contributing significantly to N pollution and to GHG emissions. In order to combat the need for such large N inputs, a better understanding of nitrogen use efficiency (NUE) and agronomic solutions that increase NUE within crops is required. The application of biostimulants derived from extracts of the brown seaweed Ascophyllum nodosum has long been accepted by growers as a sustainable crop production input. However, little is known on how Ascophyllum nodosum extracts (ANEs) can influence mechanisms of N uptake and assimilation in crops to allow reduced N application. In this work, a significant increase in nitrate accumulation in Arabidopsis thaliana 6 days after applying the novel proprietary biostimulant PSI-362 was observed. Follow-up studies in barley crops revealed that PSI-362 increases NUE by 29.85-60.26% under 75% N input in multi-year field trials. When PSI-362 was incorporated as a coating to the granular N fertilizer calcium ammonium nitrate and applied to barley crop, a coordinated stimulation of N uptake and assimilation markers was observed. A key indicator of biostimulant performance was increased nitrate content in barley shoot tissue 22 days after N fertilizer application (+17.9-72.2%), that was associated with gene upregulation of root nitrate transporters (NRT1.1, NRT2.1, and NRT1.5). Simultaneously, PSI-362 coated fertilizer enhanced nitrate reductase and glutamine synthase activities, while higher content of free amino acids, soluble protein and photosynthetic pigments was measured. These biological changes at stem elongation stage were later translated into enhanced NUE traits in harvested grain. Overall, our results support the agronomic use of this engineered ANE that allowed a reduction in N fertilizer usage while maintaining or increasing crop yield. The data suggests that it can be part of the solution for the successful implementation of mitigation policies for water quality and GHG emissions from N fertilizer usage.

Comparison of edible brown algae extracts for the inhibition of intestinal carbohydrate digestive enzymes involved in glucose release from the diet.

Type II diabetes is considered the most common metabolic disorder in the developed world and currently affects about one in ten globally. A therapeutic target for the management of type II diabetes is the inhibition of α- glucosidase, an essential enzyme located at the brush border of the small intestinal epithelium. The inhibition of α-glucosidase results in reduced digestion of carbohydrates and a decrease in postprandial blood glucose. Although pharmaceutical synthetic inhibitors are available, these are usually associated with significant gastrointestinal side effects. In the present study, the impact of inhibitors derived from edible brown algae is being investigated and compared for their effect on glycaemic control. Carbohydrate- and polyphenolic-enriched extracts derived from Ascophyllum nodosum, Fucus vesiculosus and Undaria pinnatifida were characterised and screened for their inhibitory effects on maltase and sucrase enzymes. Furthermore, enzyme kinetics and the mechanism of inhibition of maltase and sucrase were determined using linear and nonlinear regression methods. All tested extracts showed a dose-dependent inhibitory effect of α-glucosidase with IC50 values ranging from 0⋅26 to 0⋅47 mg/ml for maltase; however, the only extract that was able to inhibit sucrase activity was A. nodosum, with an IC50 value of 0⋅83 mg/ml. The present study demonstrates the mechanisms in which different brown seaweed extracts with varying composition and molecular weight distribution differentially inhibit α-glucosidase activities. The data highlight that all brown seaweed extracts are not equal in the inhibition of carbohydrate digestive enzymes involved in postprandial glycaemia.

Ascophyllum nodosum Extract (SealicitTM) Boosts Soybean Yield Through Reduction of Pod Shattering-Related Seed Loss and Enhanced Seed Production.

Soybean is one of the most valuable commercial crops because of its high protein, carbohydrate, and oil content. The land area cultivated with soybean in subtropical regions, such as Brazil, is continuously expanding, in some instances at the expense of carbon storing natural habitats. Strategies to decrease yield/seed losses and increase production efficiency are urgently required to meet global demand for soybean in a sustainable manner. Here, we evaluated the effectiveness of an Ascophyllum nodosum extract (ANE), SealicitTM, in increasing yields of different soybean varieties, in two geographical regions (Canada and Brazil). In addition, we investigated the potential of SealicitTM to reduce pod shattering at the trials in Brazil. Three different concentrations of SealicitTM were applied to pod shatter-susceptible (SS) UFUS 6901 and shatter-resistant (SR) UFUS 7415 varieties to assess their impact on pod firmness. SS variety demonstrated a significant decrease in pod shattering, which coincided with deregulation of GmPDH1.1 and GmSHAT1-5 expression, genes that determine pod dehiscence, and higher seed weight per pod. SealicitTM application to the SR variety did not significantly alter its inherent pod shatter resistance, but provided higher increases in seed yield at harvest. This yield increase maybe associated with to other yield components stimulated by the biostimulant. This work demonstrates that SealicitTM, which has previously been shown to improve pod firmness in Arabidopsis and selected commercial oilseed rape varieties through IND gene down-regulation, also has the potential to improve pod resistance and seed productivity in soybean, a member of the legume family sharing a similar strategy for seed dispersal.

Ascophyllum nodosum Extract Biostimulant Processing and Its Impact on Enhancing Heat Stress Tolerance During Tomato Fruit Set.

The application of biostimulants derived from extracts of the brown seaweed Ascophyllum nodosum has long been accepted by growers to have productivity benefits in stressed crops. The impact of the processing method of the A. nodosum biomass is also known to affect compositional and physicochemical properties. However, the identification of the mechanisms by which processing parameters of Ascophyllum nodosum extracts (ANEs) affect biostimulant performance in abiotically stressed crops is still poorly understood. In this study, we performed a comparative analysis of two carbohydrate-rich formulations derived from A. nodosum: C129, an ANE obtained at low temperatures through a gentle extraction and the novel proprietary PSI-494 extracted under high temperatures and alkaline conditions. We tested the efficiency of both ANEs in unstressed conditions as well as in mitigating long-term moderate heat stress in tomato (Lycopersicon esculentum, cv. Micro Tom) during the reproductive stage. Both ANEs showed significant effects on flower development, pollen viability, and fruit production in both conditions. However, PSI-494 significantly surpassed the heat stress tolerance effect of C129, increasing fruit number by 86% compared to untreated plants growing under heat stress conditions. The variation in efficacy was associated with different molecular mass distribution profiles of the ANEs. Specific biochemical and transcriptional changes were observed with enhanced thermotolerance. PSI-494 was characterized as an ANE formulation with lower molecular weight constituents, which was associated with an accumulation of soluble sugars, and gene transcription of protective heat shock proteins (HSPs) in heat stressed tomato flowers before fertilization. These findings suggest that specialized ANE biostimulants targeting the negative effects of periods of heat stress during the important reproductive stage can lead to significant productivity gains.

A plant biostimulant from the seaweed Ascophyllum nodosum (Sealicit) reduces podshatter and yield loss in oilseed rape through modulation of IND expression.

The yield of podded crops such as oilseed rape (OSR) is limited by evolutionary adaptations of the plants for more efficient and successful seed dispersal for survival. These plants have evolved dehiscent dry fruits that shatter along a specifically developed junction at carpel margins. A number of strategies such as pod sealants, GMOs and hybrids have been developed to mitigate the impact of pod shatter on crop yield with limited success. Plant biostimulants have been shown to influence plant development. A challenge in plant biostimulant research is elucidating the mechanisms of action. Here we have focused on understanding the effect of an Ascophyllum nodosum based biostimulant (Sealicit) on fruit development and seed dispersal trait in Arabidopsis and OSR at genetic and physiological level. The results indicate that Sealicit is affecting the expression of the major regulator of pod shattering, INDEHISCENT, as well as disrupting the auxin minimum. Both factors influence the formation of the dehiscence zone and consequently reduce pod shattering. Unravelling the mode of action of this unique biostimulant provides data to support its effectiveness in reducing pod shatter and highlights its potential for growers to increase seed yield in a number of OSR varieties.

Endochitinase 1 (Tv-ECH1) from Trichoderma virens has high subsite specificities for acetylated units when acting on chitosans.

Chitosans with defined characteristics have been shown to possess reproducible bioactivities for numerous applications. A promising approach for producing chitosans with defined degrees of polymerization (DP), degrees of acetylation (DA), and patterns of acetylation (PA) involves using chitin-modifying enzymes. One such enzyme, the chitinase Tv-ECH1 belonging to the glycoside hydrolase (GH) family 18, seems to have an important role in the biocontrol properties of the fungus Trichoderma virens, suggesting its potential in generating novel chitosans for plant health applications. In this study, the Tv-ECH1 enzyme was overexpressed in the methylotrophic yeast Pichia pastoris, yielding large amounts (up to 2mgmL-1) of purified recombinant enzyme of high activity, high purity, and high stability, making the system promising for industrial production of Tv-ECH1. The purified Tv-ECH1 chitinase displayed a wide optimal pH range from 4.5 to 6 and an optimal temperature of 37°C. Detailed subsite specificity analyses revealed high preference for acetylated residues at all four subsites analyzed (-2, -1, +1, +2), making Tv-ECH1 a promising candidate for the biotechnological production of specific chitosan oligomers and for the characterization of chitosan polymers via enzymatic fingerprinting.

Ascophyllum nodosum extract biostimulants and their role in enhancing tolerance to drought stress in tomato plants.

Global changes in climate are leading to increased occurrence and duration of drought episodes with concurrent reduction in crop yields. Expansion of the irrigated land area does not appear to be a viable solution in many regions to deliver crop productivity. The development of crop drought tolerance traits by either genetic modification or plant breeding represent the principal approaches to meeting this challenge to date. Biostimulants are an emerging category of crop management products which can enhance crop productivity under abiotic stress conditions. The ability of some biostimulant products such as Ascophyllum nodosum extracts (ANE) to enhance the tolerance of crops to drought stress has been observed by growers. The objective of this study was to investigate if different commercial ANE biostimulants provided the same tolerance to tomato plants (cv. Moneymaker) subjected to a defined drought period. A compositional characterisation of the key macromolecules of ANEs was performed. In addition, the role of ANE biostimulants in inducing changes of chlorophyll and osmolytes levels, MDA production, dehydrin isoform pattern and dehydrin gene expression levels was assessed. The three ANE biostimulants evaluated were found to provide different levels of tolerance to drought stressed tomato plants. The level of drought tolerance provided was related to changes in the concentration of osmolytes and expression of tas14 dehydrin gene. Taken together, our results highlight that despite the fact all ANE biostimulants were manufactured from the same raw material, their ability to maintain crop productivity during and after drought stress was not the same.

Comparative Transcriptome Analysis of Two Ascophyllum nodosum Extract Biostimulants: Same Seaweed but Different.

Biostimulants for crop management are gaining increased attention with continued demand for increased crop yields. Seaweed extracts represent one category of biostimulant, with Ascophyllum nodosum extracts (ANE) widely used for yield and quality enhancement. This study investigated how the composition of two ANE biostimulants (ANE A and ANE B) affects plant mRNA transcriptomes, using the model plant Arabidopsis thaliana. Using Affymetrix Ath1 microarrays, significant heterogeneity was detected between the ANE biostimulants in terms of their impacts on the mRNA transcriptome of A. thaliana plants, which accumulated significantly more biomass than untreated controls. Genes dysregulated by the ANE biostimulants are associated with a wide array of predicted biological processes, molecular functions, and subcellular distributions. ANE A dysregulated 4.47% of the transcriptome, whereas ANE B dysregulated 0.87%. The compositions of both ANEs were significantly different, with a 4-fold difference in polyphenol levels, the largest observed. The standardization of the composition of ANE biostimulants represents a challenge for providing consistent effects on plant gene expression and biostimulation.

Engineering of a fungal beta-galactosidase to remove product inhibition by galactose.

Beta-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent in most world regions. The gene encoding an acid-stable beta-galactosidase potentially suited for use as a digestive supplement was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition, a model of the enzyme structure was generated based on a Penicillium sp. beta-galactosidase crystal structure with bound beta-galactose. This led to targeted mutagenesis of an Asp(258)-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant beta-galactosidase enzyme with reduced sensitivity to inhibition by galactose (K (i) of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased K (m) (3.76 mM compared to 2.21 mM) and reduced V (max) (110.8 micromol min(-1) mg(-1) compared to 172.6 micromol min(-1) mg(-1)) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties for treatment of lactose intolerance.

A novel acid-stable, acid-active beta-galactosidase potentially suited to the alleviation of lactose intolerance.

Extracellular beta-galactosidase produced by a strain of Aspergillus niger van Tiegh was purified to homogeneity using a combination of gel filtration, ion-exchange, chromatofocusing, and hydrophobic interaction chromatographies. The enzyme displayed a temperature optimum of 65 degrees C and a low pH optimum of between 2.0 and 4.0. The monomeric glycosylated enzyme displayed a molecular mass of 129 kDa and an isoelectric point of 4.7. Protein database similarity searching using mass spectrometry-derived sequence data indicate that the enzyme shares homology with a previously sequenced A. niger beta-galactosidase. Unlike currently commercialised products, the enzyme displayed a high level of stability when exposed to simulated gastric conditions in vitro, retaining 68+/-2% of original activity levels. This acid-stable, acid-active beta-galactosidase was formulated, along with a neutral beta-galactosidase from Kluyveromyces marxianus DSM5418, in a novel two-segment capsule system designed to ensure delivery of enzymes of appropriate physicochemical properties to both stomach and small intestine. When subjected to simulated full digestive tract conditions, the twin lactase-containing capsule hydrolyzed, per unit activity, some 3.5-fold more lactose than did the commercial supplemental enzyme. The acid-stable, acid-active enzyme, along with the novel two-segment delivery system, may prove beneficial in the more effective treatment of lactose intolerance.