Soluble Protein Research Articles

CEAM is a mitochondrial-localized, amyloid-like motif-containing microprotein expressed in human cardiomyocytes

Microproteins synthesized through non-canonical translation pathways are frequently found within mitochondria. However, the functional significance of these mitochondria-localized microproteins in energy-intensive organs such as the heart remains largely unexplored. In this study, we demonstrate that the long non-coding RNA CD63-AS1 encodes a mitochondrial microprotein. Notably, in ribosome profiling data of human hearts, there is a positive correlation between the expression of CD63-AS1 and genes associated with cardiomyopathy. We have termed this microprotein CEAM (CD63-AS1 encoded amyloid-like motif containing microprotein), reflecting its sequence characteristics. Our biochemical assays show that CEAM forms protease-resistant aggregates within mitochondria, whereas deletion of the amyloid-like motif transforms CEAM into a soluble cytosolic protein. Overexpression of CEAM triggers mitochondrial stress responses and adversely affect mitochondrial bioenergetics in cultured cardiomyocytes. In turn, the expression of CEAM is reciprocally inhibited by the activation of mitochondrial stresses induced by oligomycin. When expressed in mouse hearts via adeno-associated virus, CEAM impairs cardiac function. However, under conditions of pressure overload-induced cardiac hypertrophy, CEAM expression appears to offer a protective benefit and mitigates the expression of genes associated with cardiac remodeling, presumably through a mechanism that suppresses stress-induced translation reprogramming. Collectively, our study uncovers a hitherto unexplored amyloid-like microprotein expressed in the human cardiomyocytes, offering novel insights into myocardial hypertrophy pathophysiology.

Coarse-Graining the Recognition of a Glycolipid by the C-Type Lectin Mincle Receptor.

Macrophage inducible Ca2+-dependent lectin (Mincle) receptor recognizes Mycobacterium tuberculosis glycolipids to trigger an immune response. This host membrane receptor is thus a key player in the modulation of the immune response to infection by M. tuberculosis and has emerged as a promising target for the development of new vaccines against tuberculosis. The recent development of the Martini 3 force field for coarse-grained (CG) molecular modeling allows the study of interactions of soluble proteins with small ligands which was not typically modeled well with the previous Martini 2 model. Here, we present a refined approach detailing a protocol for modeling interactions between a glycolipid and its receptor at a CG level using the Martini 3 force field. Using this approach, we studied Mincle and identified critical parameters governing ligand recognition, such as loop flexibility and the regulation of hydrophobic groove formation by calcium ions. In addition, we assessed ligand affinity using free energy perturbation calculations. Our results offer mechanistic insight into the interactions between Mincle and glycolipids, providing a basis for the rational design of molecules targeting this type of membrane receptors.

Demystifying the nutritional and anti-nutritional genetic divergence of Pakistani chickpea (Cicer arietinum L.) genetic resource via multivariate approaches.

Chickpeas are a highly versatile functional food legume that possesses the capacity to boost human health and has the potential to alleviate malnutrition-related deficiencies. To investigate whole seed-based nutritional and anti-nutritional composition, a set of 90 chickpea genotypes (66 desi and 24 kabuli) was collected from different research organizations in Pakistan. Significant variation (Tukey HSD test, p < 0.05) was perceived among genotypes for traits under investigation. The genotypes, with maximum total soluble proteins (TSPs) (34.92%), crude proteins (CPs) (30.13%), and reducing sugars (17.33 mg/g s. wt.), i.e., Punjab-2000 (desi); total free amino acids (TFAs) (3.34 g/100 g DW), i.e., Wild Hybrid-15 (desi), albumins (227.67 mg/g s. wt.), i.e., Sheenghar-2000 (desi); globulins (720 g s. wt.), i.e., ICCV-96030 (desi); salt-soluble proteins (200 mg/g s. wt.), i.e., ILWC-247 (desi); total soluble sugars (TSSs) (102.63 mg/g s. wt.), i.e., CM1051/11 (desi); non-reducing sugars (95.28 mg/g s. wt.), i.e., NIAB-CH2016 (desi); starch content (83.69%), i.e., CH55/09 (kabuli); and the genotypes with least value of anti-nutritional factors glutelin (3.33 mg/g s. wt.), i.e., Wild Hybrid-9 (desi); hordein (1.38 mg/g s. wt.), i.e., Noor-2013 (kabuli); tannins (5,425 uM/g s. wt.), i.e., Wild Hybrid-1 (desi); and phytic acid (PA) (0.18 mg/g s. wt.), i.e., Bhakhar-2011 (desi), could be the promising genotypes to formulate health-promoting plant-based food products. Data were also analyzed for principal component analysis (PCA), correlation, and agglomerative hierarchical clustering. PC-1 revealed the highest contribution (20.83%) toward cumulative variability, and maximum positive factor loading was delivered by TSSs (0.85) followed by starch content (0.729). Genotypes were grouped into three distinct clusters based on high average values of traits under investigation. Cluster I encompassed genotypes with a high mean value of CP content, albumins, hordein, and glutelin; Cluster II encompassed genotypes with a high mean value of TSPs, TSSs, non-reducing sugars, globulins, salt-soluble sugars, starch, and TFAs; Cluster III encompassed genotypes with high tannins, reducing sugars, and PA. Identified desi and kabuli genotypes exhibiting superior seed quality traits and minimal anti-nutritional factors can be used in chickpea breeding programs aimed at improving seed nutritional quality in future breeding lines.

How to isolate channel-forming membrane proteins using the E. coli expression system.

The recombinant expression, isolation and characterization of pore-forming proteins is one of the most commonly used strategies for understanding the permeability properties of the biological membrane into which they are embedded. This protocol describes how to quantify the expression of your protein of interest and use this information to optimize its production using the Escherichia coli strain BL21Gold(de3)ΔABCF. It explains with a step-by-step approach how to separate the bacterial compartments according to their solubility and how to extract your protein of interest in its native conformation using detergent solutions. Finally, it describes how to improve its purity via ion-exchange chromatography and insert the purified porins into outer membrane vesicles, from which they can be copurified. The protocol is simpler and less empirical than those described for most channel-forming membrane proteins and also provides a solid foundation for the isolation of soluble proteins. Several parameters can be optimized on a case-by-case basis: expression time and temperature, concentration of the inducer, nature and concentration of the detergent, incubation time and temperature, pH and ionic strength of the purification buffers. This protocol is effective with prokaryotic channel-forming membrane proteins and can be employed for the production of pore-forming proteins from chloroplasts, mitochondria or eukaryotes in general. With minor optimization, this protocol can be adapted for the isolation of receptors, carrier, pumps or any other membrane-active proteins.

Measurement of absolute abundance of crystallins in human and αA N101D transgenic mouse lenses using 15N-labeled crystallin standards

Stable isotope labeled standards of all major human lens crystallins were created to measure the abundance of lens endogenous crystallins from birth to adulthood. All major human crystallins (αA, αB, βA2, βA3/A1, βA4, βB1, βB2, βB3, γA, γB, γC, γD, γS) were cloned with N-terminal 6 x His tagged SUMO for ease of purification and the ability to generate natural N-termini by SUMO protease cleavage when producing crystallins for structure/function studies. They were then expressed in 15N-enriched media, quantified by mass spectrometry, and mixed in proportions found in young human lens to act as an artificial lens standard. The absolute quantification method was tested using soluble protein from 5-day, 23-day, 18-month, and 18-year-old human lenses spiked with the 15N artificial lens standard. Proteins were trypsinized, relative ratios of light and heavy labeled peptides determined using high-resolution precursor and data independent MS2 scans, and data analysis performed using Skyline software. Crystallin abundances were measured in both human donor lenses and in transgenic mouse αA N101D cataract lenses. Technical replicates of human crystallin abundance measurements were performed with average coefficients of variation of approximately 2% across all 13 crystallins. αA crystallin comprised 27% of the soluble protein of 5-day-old lens and decreased to 16% by 18-years of age. Over this time period αB increased from 6% to 9% and the αA/αB ratio decreased from 4.5/1 to 2/1. γS-crystallin also increased nearly 2-fold from 7% to 12%, becoming the 3rd most abundant protein in adult lens, while βB1 increased from 14% to 20%, becoming the most abundant crystallin of adult lens. Minor crystallins βA2, βB3, and γA comprised only about 1% each of the newborn lens soluble protein, and their abundance dropped precipitously by adulthood. While 9 of the SUMO tagged crystallins were useful for purification of crystallins for structural studies, γA, γB, γC, and γD were resistant to cleavage by SUMO protease. The abundance of WT and N101D human αA in transgenic mouse lenses was approximately 40-fold lower than endogenous mouse αA, but the deamidation mimic human αA N101D was less soluble than human WT αA. The high content of αA and the transient abundance of βA2, βB3, and γA in young lens suggest these crystallins play a role in early lens development and growth. βB1 becoming the most abundant crystallin may result from its role in promoting higher order β-crystallin oligomerization in mature lens. The full set of human crystallin expression vectors in the Addgene repository should be a useful resource for future crystallin studies. 15N labeling of these crystallins will be useful to accurately quantify crystallins in lens anatomic regions, as well as measure the composition of insoluble light scattering crystallin aggregates. The standards will also be useful to measure the abundance of crystallins expressed in transgenic animal models.

Characterization and quantification of peptaibol produced by novel Trichoderma spp: Harnessing their potential to mitigate moisture stress through enhanced biochemical and physiological responses in black pepper (Piper nigrum L.).

Trichoderma spp. is primarily applied to manage biotic stresses in plants. Still, they also can mitigate abiotic stresses by the stimulation of antioxidative protective mechanisms and enhanced synthesis of secondary metabolites. The study optimized the conditions to enhance peptaibol production by novel Trichoderma spp, characterized and quantified peptaibol- alamethicin using HPLC and LC MS-MS. The present study investigated these isolates efficacy in enhancing growth and the associated physio-biochemical changes in black pepper plants under moisture stress. Under in vitro conditions, out of 51 isolates studied, six isolatesviz., T. asperellum (IISR NAIMCC 0049), T. erinaceum (IISR APT1), T. harzianum (IISR APT2), T. harzianum (IISR KL3), T. lixii (IISR KA15) and T. asperellum (IISR TN3) showed tolerance to low moisture levels (5, 10 and 20%) and higher temperatures (35 and 40°C). In vivo evaluation on black pepper plants maintained under four different moisture levels (Field capacity [FC]; 75%, 50%, and 25%) showed that the plants inoculated with Trichoderma accumulated greater quantities of secondary metabolites viz., proline, phenols, MDA and soluble proteins at low moisture levels (50% and 25% FC). In the present study, plants inoculated with T. asperellum and T.harzianum showed significantly increased growth compared to uninoculated plants. The shortlisted Trichoderma isolates exhibited differences in peptaibol production and indicated that the peptide might be the key factor for their efficiency as biocontrol agents. The present study also demonstrated that Trichoderma isolates T. harzianum and T. asperellum (IISR APT2 & NAIMCC 0049) enhanced the drought-tolerant capabilities of black pepper by improving plant growth and secondary metabolite production.

Multiple Regulatory Mechanisms Synergistically Control the Soluble Expression of CsCE for Enhanced Enzymatic Productivity of Lactulose in E. coli.

The limited expression of cellobiose 2-epimerase poses a significant constraint on the industrial enzymatic production of lactulose. Extensive modifications to the expression cassette offer a means to enhance the yield of recombinant proteins. In this study, an integrated strategy, combining individual and collaborative approaches, is proposed to fine-tune each stage of the CsCE overexpression program. This strategy involves the multidimensional integration of standardized genetic elements at various levels, including transcription, translation, folding, and three-dimensional structure. The volumetric activity of the final recombinant strain was markedly increased by 12-fold compared to the wild-type strain, reaching 2260.62 U/L. The protein expression in the newly developed high-yield recombinant strain exhibited a significant enhancement, with a higher proportion of soluble protein compared to that of inclusion bodies. Our findings offer insights into the multifaceted synergistic regulation of protein expression processes, holding promising implications for the production of heterologous recombinant proteins.

Flour Treatments Affect Gluten Protein Extractability, Secondary Structure, and Antibody Reactivity

Commercial Brazilian wheat flour was subjected to extrusion, oven, and microwave treatments. The solubility, monomeric and polymeric proteins, and the glutenin and gliadin profiles of the gluten were analyzed. In addition, in vitro digestibility and response against potential celiac disease immune-stimulatory epitopes were investigated. All treatments resulted in low solubility of the polymeric and monomeric proteins. The amounts of insoluble proteins increased from 5.6% in control flour to approximately 10% for all (treatments), whereas soluble proteins decreased from 6.5% to less than 0.5% post treatment. In addition, the treatments affected glutenin and gliadin profiles. The amount of α/β-gliadin extracted decreased after all treatments, while that of γ-gliadin was unaffected. Finally, the potential celiac disease immune stimulatory epitopes decreased in oven and microwave treatment using the G12 ELISA, but no change was observed using the R5 antibody. However, the alteration of the gluten structure and complexity was not sufficient to render a product safe for consumption for individuals with celiac disease; the number of potential celiac disease immune-stimulatory epitopes remained high.

Inflammatory proteins associated with Alzheimer’s disease reduced by a GLP1 receptor agonist: a post hoc analysis of the EXSCEL randomized placebo controlled trial

BackgroundGlucagon-like peptide-1 receptor agonists are a viable option for the prevention of Alzheimer’s disease (AD) but the mechanisms of this potential disease modifying action are unclear. We investigated the effects of once-weekly exenatide (EQW) on AD associated proteomic clusters.MethodsThe Exenatide Study of Cardiovascular Event Lowering study compared the cardiovascular effects of EQW 2 mg with placebo in 13,752 people with type 2 diabetes mellitus. 4,979 proteins were measured (Somascan V0.4) on baseline and 1-year plasma samples of 3,973 participants. C-reactive protein (CRP), ficolin-2 (FCN2), plasminogen activator inhibitor 1 (PAI-1), soluble vascular cell adhesion protein 1 (sVCAM1) and 4 protein clusters were tested in multivariable mixed models.ResultsEQW affected FCN2 (Cohen’s d -0.019), PAI-1 (Cohen’s d -0.033), sVCAM-1 (Cohen’s d 0.035) and a cytokine-cytokine cluster (Cohen’s d 0.037) significantly compared with placebo. These effects were sustained in individuals over the age of 65 but not in those under 65.ConclusionsEQW treatment was associated with significant change in inflammatory proteins associated with AD.Trial RegistrationEXSCEL is registered on ClinicalTrials.gov: NCT01144338 on 10th of June 2010.

Effects of Steel Slag Used as Substrate on the Growth of Hydrangea macrophylla Cuttings

Steel slag is an industrial solid waste produced during the steelmaking process. To explore the application of steel slag in the agricultural field, the present experiment was carried out to study the effect of substrates with different contents of steel slag on the growth of Hydrangea macrophylla cuttings. The conventional substrate (perlite: vermiculite: peat = 1:1:1) was used as the control (CK), and the treatments were designed as T1 (steel slag: perlite: vermiculite: peat = 1:3:3:3, v/v/v/v), T2 (steel slag: perlite: vermiculite: peat = 1:2:2:2, v/v/v/v), T3 (steel slag: perlite: vermiculite: peat = 1:1:1:1, v/v/v/v), and T4 (steel slag: perlite: vermiculite: peat = 1:0:0:0, v/v/v/v). The results showed that the addition of steel slag significantly increased the substrate’s bulk density, EC, and pH and improved its water retention capacity to a certain extent. There were significant differences among different treatments in morphological indicators, root growth and development, and physiological and biochemical characteristics of cutting seedlings. All traits, including plant height, fresh weight, dry weight, root length, root surface area, root volume, the number of root tips, root activity, and soluble protein content of seedlings grown in T3 were significantly higher than those in other substrates. The results indicated that the appropriate addition of steel slag is helpful to hydrangea cuttings’ growth, and the optimal mixing ratio is steel slag: perlite: vermiculite: peat = 1:1:1:1 (v/v/v/v). This is a significant innovation in applying steel slag in agricultural production.

Effects of Planting Density and Nitrogen Fertilization on Growth Traits and Leaf and Wood Characteristics of Three Poplar Clones

A comprehension of the effects planting density and nitrogen (N) fertilization have on the physiological and morphological characteristics of trees is critical for optimizing the require size and characteristics of wood products. We evaluated the growth traits and the leaf and wood characteristics of three clone poplars including Populus simonii × P. nigra ‘Xiaohei’, ‘Xiaohei-14’ and ‘Bailin-3’ under five planting densities (1666, 1111, 833, 666, and 555 tree ha−1) and four N fertilization rates (0, 100, 160, and 220 g tree−1 year−1). The results show that the clone type significantly affected all observed indicators, while planting density and N fertilization treatments had a significant effect on growth traits and leaf characteristics, but not on wood characteristics. Specifically, the clone ‘Bailin-3’ exhibited the largest annual increments in tree height and diameter at breast height (DBH), leaf width, N content, and soluble protein content. A decrease in initial planting density (from 1666 to 555 tree ha−1) led to an increased annual incremental tree height and DBH, regardless of clone type and N fertilization treatment. N fertilization treatment significantly impacted the annual increment in DBH, but not that of tree height. Further, the annual increments in tree height and DBH were positively correlated with leaf width, N content, chlorophyll content, and soluble protein content, and negatively correlated with hemicellulose content. In addition, the chlorophyll and soluble protein contents were identified as the most reliable predictors of the annual increments in tree height and DBH. Our results demonstrate the clone ‘Bailin-3’ with 555 tree ha−1 under 160 g N tree−1 yr−1 showed superior growth traits and leaf characteristics. Thus, it is recommended for future poplar silviculture of larger diameter timber production at similar sites. The results contribute to understanding of the effects of planting density and fertilization on the growth traits and the leaf and wood characteristics of three poplar clones, offering valuable guidance for the sustainable development and long-term productivity of poplar plantations.

Insoluble proteomics analysis of acute intracranial large vessel occlusive thrombus

BackgroundAcute large vessel occlusion (LVO) stroke is highly prevalent and severe. Despite thrombolytic therapy, many patients experience substantial complications. Understanding the origins, constituents, and pathologic processes involved in thrombus formation in acute intracranial large artery occlusion is crucial. ObjectivesTo identify the characteristics of insoluble proteins from different sources of cerebral thrombus. MethodsThis study included 13 patients with cardiogenic embolic (CE) thrombus and 15 with large artery atherosclerotic (LAA) thrombus. High-performance liquid chromatography and liquid chromatography-tandem mass spectrometry were used to analyze insoluble proteins in thrombi. Bioinformatics analyses explored differential proteins and associated functional pathways. Least absolute shrinkage and selection operator and random forest identified biomarkers for diagnosing thrombus sources, validated by parallel reaction monitoring. ResultsWe constructed an insoluble protein atlas of cerebral thrombi, identifying 6975 insoluble proteins, including 143 extracellular matrix (ECM)-related proteins. The enrichment pathways considerably varied between thrombi from different sources. Inflammation-related pathways, such as acute inflammatory response, along with ECM-related pathways such as laminin interactions, were notably upregulated in LAA compared with CE. Additionally, 2 biomarkers (IDH2 and HSPG2) exhibited strong diagnostic performance (area under the curve = 1) and robustness. ConclusionIn the insoluble proteomics of thrombus, we highlighted the crucial roles of immune responses and ECM proteins in thrombus formation, providing new insights into its mechanisms and potential drug development. Additionally, we identified 2 biomarkers that offer new methods for determining thrombus sources in patients with LVO.

Optimization of extracellular polysaccharides (EPS) production by Stenotrophomonas rhizophila JC1 and its protective effect on alfalfa under Pb2+ stress

Stenotrophomonas rhizophila JC1 and its extracellular polysaccharides (EPS) have been shown to effectively adsorb heavy metals in previous studies. The fermentation conditions of EPS by S. rhizophila JC1 were optimized using the Box-Behnken design (BBD). The composition, structural characteristics, and heavy metal adsorption capacity of EPS were systematically evaluated. The alleviation mechanism of Pb2+ stress on alfalfa was investigated through EPS inoculation. The maximum EPS yield reached 0.313 %. EPS consisted of glucose, glucosamine, galactose, and mannose in a molar ratio of 12.20:1:22.29:1.68. EPS also contained four distinct polymers with molecular weights of 623,683.71 Da, 144,072.27 Da, 105,892.21 Da, and 51,094.79 Da. The adsorption processes conformed to the pseudo-second-order model and Langmuir isotherm model. High Pb2+ concentrations significantly reduced germination percentage, germinative force, root length, fresh weight, and soluble protein, inhibited photosynthesis, exacerbated oxidative stress, and caused damage to the antioxidant system, thereby inhibiting seedling growth. EPS at low concentrations can promote alfalfa seed germination and mitigate Pb2+ stress by reducing the aforementioned damage. This study highlights the potential of EPS in soil remediation and enhancing plant resistance to heavy metal stress.

Drought and rewatering practices improve adaptability of seedling maize to drought stress by a super-compensate effect

Periodic drought adversely affects the growth and yield of summer crops in the Huang-Huai-Hai Plain. Drought-rewatering practice as one of the important agronomic measures to improve crop drought resistance. A field experiment was conducted to investigate practice physiological, biochemical, and molecular responses of maize seedling after two rounds of repeated drought and rewatering treatments. The results demonstrated that rewatering following repeated drought events had a compensatory effect on the photosynthetic rate (Pn) and on osmotic and antioxidant regulation. Specifically, the Pn and stomatal conductance (Gs) increased by 10.12% and 5.61%, respectively, compared to the control (CK) during the second round of treatment. Additionally, soluble protein (sPro) and proline (Pro) content rose significantly, with increases of 26.12% and 343.49% observed on day 5 of the second round, leading to a gradual reduction in leaf water content and osmosis. Following drought exposure, the activities of superoxide dismutase (SOD) and peroxidase (POD) contributed to the decreased levels of malondialdehyde (MDA), with both enzymes recovering during rewatering. In contrast, plant height, leaf area, and biomass were significantly reduced in the CK group. Notably, root length increased by 21.05% after the drought-rewatering practice, enhancing the maize seedlings' ability to adapt to drought stress. Overall, maize seedlings exhibited enhanced adaptability to drought conditions following two cycles of drought-rewatering treatments.

The effect of several growth regulators and biostimulant on biochemical and physiological changes in acclimation of micropropagated Echinacea purpurea Moench. ‘Raspberry Truffle’

Micropropagation is currently one of the primary methods for plant propagation, known for its efficiency in producing disease-free and vigorous plants. However, the final stage of this—acclimatization, is critical due to the transfer from a controlled in vitro environment to an external one. To reduce mortality and alleviate acclimation stress, plant growth regulators (PGRs) or biostimulants can be employed. This study investigated the effects of exogenously sprayed PGRs: 0.001 mg L−1 abscisic acid (ABA), 0.001 mL L−1 brassinolide (BL), 0.001 mL L−1 24-epibrassinolide (24-epiBL), and 0.3 mL L−1 biostimulant Goteo on the physiological and biochemical responses of Echinacea purpurea ‘Raspberry Truffle’ plantlets during the acclimation process. The effects of treatments at various acclimation stages on chlorophyll (chl) and carotenoids content, hydrogen peroxide (H2O2), catalase (CAT), malondialdehyde (MDA), free amino acids, soluble proteins, total soluble sugar and reduced soluble sugars were tested in this research. The results confirmed changes in biochemical parameters, including an increase in chlorophyll and carotenoids in the acclimatization period where the highest level obtained by BL spraying. A decline in stomatal conductance was also observed, where ABA influenced the most on drop. It was also recorded the decrease in H2O2 and MDA concentration. CAT activity increased, especially with biostimulant treatment. We recorded an increase in total soluble proteins along acclimatization. Goteo affected the most on morphology parameters, ABA, BL and 24-epiBL increased acclimatization efficiency. Our studies indicate that potentially the most effective substances in the acclimatization of E. purpurea ‘Raspberry Truffle’ are brassinosteroids and ABA.

Effect of microwave-puffed on Auricularia auricula polysaccharide and probiotic fermentation on its biotransformation and quality characteristics during storage period

In this study, probiotics with superior fermentation performance were screened, and the mixed-bacteria fermentation was carried out with Auricularia auricula treated with microwave-puffed process as fermentation substrate, and the changes in nutritional quality under different storage conditions were investigated. The results showed that the acid and bile salt resistance of Lactiplantibacillus plantarum 21,801 and 21,805 reached 95 % and 75 % respectively, and the intestinal adhesion was superior; microwave puffing treatment had the highest retention rate of A. auricula polysaccharides and the lowest loss of polyphenols, and no effect on soluble protein. Mixed bacterial fermentation significantly increased the total polyphenols and total flavonoids of A. auricula (p < 0.05), and the DPPH and ABTS radical scavenging reached 48.31 % and 73.21 % respectively. Furthermore, the viable counts, DPPH radical scavenging, color, and sensory quality of fermented A. auricula remained stable when stored at 4 °C. In contrast, when stored at 25 °C for 7 days, the taste was unfavorable, undesirable odor and spoilage occurred; by 21 days, DPPH clearance rate dropped below 40 % and color changed significantly (△E > 2). In conclusion, the probiotic mixed fermentation and storage conditions had a significant effect on the biometabolic transformation of macromolecules and other substances in A. auricula.

Production of Norovirus VLPs of the Nine Representative Genotypes Widely Distributed in Japan using the Silkworm-Baculovirus Expression Vector System

Norovirus (NoV) is one of the major causes of acute viral gastroenteritis in humans. Genetic variation is abundant, and prevalent genotypes vary from year to year and region to region. Since NoVs are difficult to amplify in cultured cells, genome RNA-free virus-like particles (VLPs) that mimic the capsid structure of the virus are promising vaccine candidates for the prevention of NoVs infection, and the development of multivalent VLP vaccines is required to prevent NoV infection in a wide range of genotypes. In this study, we attempted to produce NoV VLPs of the top nine genotypes that have a history of epidemics in Japan using the silkworm-baculovirus expression vector system (silkworm-BEVS), which has a proven track record in the mass production of recombinant proteins. In silkworm pupae infected with recombinant baculoviruses constructed to express VP1s, the major protein that forms VLP, the NoV VP1 protein was expressed in large amounts. Most genotypes of VP1 accumulated in the cytoplasm as soluble proteins, but solubility was reduced for that of two genotypes. VP1s of five genotypes could be purified in large quantities (>0.9mg per pupa) by a two-step purification process, and gel filtration chromatography analysis confirmed the formation of VLPs. This study demonstrates the utility of silkworm-BEVS in producing NoV VLPs of multiple genotypes and provides the basis for the development of a multivalent vaccine against genetically diverse NoV infections.

Structural basis of selective beta-carotene binding by a soluble protein

β-carotene (BCR) is the most abundant carotenoid, a colorant, antioxidant, and provitamin A. The extreme hydrophobicity of this hydrocarbon requires special mechanisms for distribution in aqueous media, including water-soluble carotenoproteins. However, all known carotenoproteins prefer oxygenated carotenoids and bind BCR inefficiently. Here, we present the crystal structure of the BCR-binding protein (BBP) from gregarious male locusts, which is responsible for their vivid yellow body coloration, in complex with its natural ligand, BCR. BBP forms an antiparallel tubular homodimer with α/β-wrap folded monomers, each forming a hydrophobic 47Å long, coaxial tunnel that opens outward and is occupied by one s-cisC6-C7, all-trans BCR molecule. In the BCR absence, BBP accepts a range of xanthophylls, with reduced efficiency depending on the position and number of oxygen atoms, but rejects lycopene. The structure captures a pigment complex with a Takeout 1 protein and inspires potential applications of BBP as a BCR solubilizer.

Multi-functional PGPR Serratia liquefaciens confers enhanced resistance to lead stress and bacterial blight in soybean (Glycine max L.)

Lead toxicity and bacterial blight disease caused by Pseudomonas savastanoi pv. glycinea have destructive impacts on soybean growth and productivity. Plant growth-promoting rhizobacteria have been used as an eco-friendly approach for augmenting crop growth and stress resistance. The current study investigated the efficacy of Serratia liquefaciens ZM6 strain in enhancing soybean resistance to lead (Pb) stress and bacterial blight. Two pot experiments were performed. In the first pot experiment, soybean plants were inoculated with S. liquefaciens ZM6 and grown under variable Pb stress levels (0, 200 and 400µM of Pb(NO3)2). In the second experiment, S. liquefaciens-inoculated soybean plants were infected with P. savastanoi pv. glycinea, and disease severity was assessed two weeks post infection. The results revealed that S. liquefaciens strain resisted Pb stress up to 400µM Pb(NO3)2 and exhibited the highest levels of solubilized phosphate, solubilized zinc, siderophore, indole acetic acid, exopolysaccharide, trehalose and antioxidant enzymes at 400µM Pb compared to the other treatments. Moreover, Pb stress (200 and 400µM) significantly decreased the growth, yield, nutrient uptake, gas exchange, and contents of chlorophyll, soluble proteins, sugars, and phenolics of soybean plants. Pb stress also induced the levels of proline, glycine betaine, Pb, oxidative stress markers, antioxidant enzymes, ascorbate, glutathione and expression of stress-responsive genes (CAT, APX, POD, Fe-SOD, CHS7, CHI1A, PAL, IFS2, P5CS and WRKY54) in soybean plants. On the other hand, S. liquefaciens application markedly boosted the growth, yield and levels of nutrients, gas-exchange, chlorophyll, osmolytes, antioxidant enzymes and expression of stress-tolerant genes of Pb-stressed soybean plants. The bacterial inoculation significantly diminished oxidative stress indicators and Pb content in stressed plants. Inoculation of soybean plants with S. liquefaciens also caused significant reductions in blight disease symptoms in P. savastanoi pv. glycinea-infected plants, indicating the efficiency of this strain in controlling harmful blight disease. Overall, this study demonstrated S. liquefaciens ZM6 effectiveness in enhancing soybean resistance to Pb stress and bacterial blight.

Biochar solutions: Slow and fast pyrolysis effects on chromium stress in rapeseed roots

Chromium (Cr) contamination in agricultural soils, largely due to industrial activities, poses a significant threat to plant growth and productivity. This study examines the effects of Cr stress at concentrations of 100 and 200 mg of K2Cr2O7 per kg soil on rapeseed (Brassica napus) roots and evaluates the mitigating potential of biochar. Biochar, produced through both slow and fast pyrolysis and applied at 30 g per kg soil, was investigated for its ability to neutralize Cr toxicity. Our findings indicate that Cr stress significantly decreased the growth and physiological functions of rapeseed roots. However, biochar application improved soil pH, cation exchange capacity, and the uptake of essential nutrients such as nitrogen, phosphorus, potassium, calcium, and magnesium. Additionally, biochar enhanced the production of osmotic regulators like glycine betaine and soluble proteins, as well as indole acetic acid, promoting better root growth and water uptake under Cr stress. Notably, biochar reduced Cr availability and absorption in rapeseed roots, leading to lower levels of stress-related hormones such as abscisic acid, salicylic acid, and jasmonic acid. Among the biochars tested, slow pyrolysis biochar was more effective than fast pyrolysis biochar in mitigating Cr toxicity. These results highlight the potential of slow pyrolysis biochar as a sustainable strategy to alleviate Cr pollution and enhance plant resilience in contaminated soils.