Ancestral sequence reconstruction (ASR) offers a revolutionary approach to resurrect functional proteins, yet its potential in transporter engineering remains underexplored. Here, we pioneered the application of ASR to reconstructing ancestral xylose transporters, addressing the persistent challenge of glucose-mediated inhibition of xylose uptake in Saccharomyces cerevisiae during xylose co-fermentation. Through rigorous ASR analysis, we reconstructed ancestral xylose transporters (Xt) and selected two candidates—Xt3 (approximately 140 million years old) and Xt7 (approximately 40 million years old)—based on their phylogenetic positioning, degree of sequence divergence from extant homologs, and predicted structural integrity. Functional characterization demonstrated that both Xt3 and Xt7 significantly enhance xylose uptake efficiency and mitigate glucose-induced repression. In fermentation experiments with mixed sugars (40 g/L xylose and 40 g/L glucose) within 72 h, recombinant S. cerevisiae expressing Xt3 achieved 22.75 g/L xylose consumption, surpassing the benchmark N326F^Xltr1p (16.22 g/L) by 40.27% and outperforming Xt7 (21.36 g/L) by 6.51%, highlighting Xt3 as the most efficient transporter. Molecular docking suggested a potentially more favorable binding mode for xylose in the ancestral transporters (binding affinity: −3.68 kcal/mol for Xt3 vs. −3.15 kcal/mol for N326F^Xltr1p). Molecular dynamics simulations further demonstrated that the ancestral transporters formed complexes with xylose that exhibited faster convergence to a stable state and maintained significantly greater conformational stability throughout the simulation compared to the N326F^Xltr1p complex. These computational insights provide a plausible structural basis for their enhanced performance. This work contributes to the advancement of lignocellulosic biorefinery technology and provides a practical reference for resurrecting other valuable proteins using ASR’.

Natural exogenous additives (EA) suitable for the tobacco fermentation need to be developed to enhance the fermentation quality and economic value of low-grade cigar tobacco leaves (CTLs). This study analyzed the impacts of three compound Chinese herbal medicine (CHM) on metabolites and microorganisms during CTLs fermentation. The results manifested that EA facilitated the degradation of total sugar, starch and protein, while enhancing the accumulation of reducing sugar in CTLs. Furthermore, EA raised contents of free amino acids (FAAs), while Asp, Glu, Ser and His were found to be key differential FAAs of CTLs. During fermentation, the total contents of volatile flavor components (VFCs) initially increased and then declined. Furthermore, EA contributed to more harmonious compositions of VFCs by promoting the formation of neophytadiene, ketones, esters and aldehydes, as well as facilitating nicotine degradation. According to variable importance in the projection (VIP) > 1 and odor activity value (OAV) > 1, 7 key differential VFCs were identified. EA enhanced positive microbial interactions and led to a more stable and coordinated symbiotic network. Linear discriminant analysis effect size (LEfSe) identified 9 genera as differentially dominant microorganisms in CTLs, which were closely associated with chemical compositions and key differential flavor metabolites. In addition, EA promoted cigar tobacco characteristics (CTCs) by altering bacterial alpha diversity and influencing the assembly of dominant microbial communities. Overall, this study offered theoretical insights into the innovative applications of CHM in CTLs fermentation, and presented new perspectives for enhancing CTLs quality and customizing flavor profiles.

Escherichia coli, a bacterium indicating improper hygiene practices during food production, is commonly found in the intestines of humans and animals, while Salmonella spp. are dangerous bacteria that cause typhoid fever and severe diarrhea. These pathogens have been found in fresh vegetables. This study investigated how the vegetable surface characteristics influenced bacterial adhesion. The reduction of bacteria during the washing process was assessed using different concentrations and types of chemicals. The relationships between variables obtained from image analysis techniques and bacterial adhesion on vegetable surfaces were also evaluated. The most effective way to inhibit bacteria was by washing with 2.0% lactic acid, with bacterial reduction from an initial concentration of 8.74 to 2.92 log CFU/m². Pearson’s correlation with the highest r value was surface area (A) with values ranging from 0.764 to 0.993, followed by surface roughness (R) with values between 0.019 and 0.986, and Fractal dimension (FD) with values between − 0.510 and − 0.992. The correlation between A and the number of bacteria (E. coli and Salmonella) was the highest, with surface area influencing bacterial adhesion to the vegetable surface. Greater surface roughness was associated with a higher initial bacterial load, making the A value a good predictor of changes in bacteria during washing with organic acids at various concentrations.

Synthetic chemical seed treatments, while effective, often raise significant environmental and health concerns. These concerns stem from the use of hazardous chemicals such as fungicides and insecticides that, besides posing risks to workers, have broader environmental impacts. These hazardous chemicals can leach into the soil and water systems, disrupting ecosystems, harming beneficial organisms, and entering the food chain. Agro-industrial byproducts/wastes (AIBWs) represent an abundant, environmentally friendly resource with potential for seed treatments. We focused on AIBWs that are produced in enormous amounts and do not pose potential hazards since they are commonly used to feed animals as well as food additives for humans, including wheat bran (WB), wine pomace (WP), and brewer’s spent grain (BSG). We investigated the effects of imbibing wheat seeds in water-soluble extracts of AIBWs or coating seeds with a biopolymer supplemented with AIBW substances on wheat growth and reproduction. As controls, we used water-soaked (WS) and non-soaked (NS) seeds, as well as chemically Celest Top-coated seeds. Petri dish assays showed that seeds imbibed in AIBW extracts exhibited enhanced post-germination growth as compared to NS seeds. Thus, while 81% of NS seedlings produced up to 3 seminal roots (SRs), 84% of WB and 64% of Celest Top seedlings produced 4 and 5 SRs. Net-house experiments revealed that Celest Top and AIBW extracts had a positive effect on reproduction as compared to NS, displaying 17.4%, 14.5%, 30.3%, and 34.3% increases in grain weight per spike in Celest Top, WB, GP, and WP, respectively. Metabolic analysis of seeds derived from treated plants revealed variation in metabolite profiles with a notable increase in the amino acid tryptophan. We utilized the nature-sourced polysaccharide carboxymethylcellulose (CMC) to coat seeds with AIBW substances derived from GP, referred to as CMC-GP. The results indicated that CMC-GP and Celest Top enhanced root growth, displaying 2- and 1.5-fold increases in fresh and dry weight, respectively, as compared to NS and CMC-coated seeds. Thus, AIBWs appear to provide cost-effective, eco-friendly alternatives to the hazardous chemical seed coatings, whether applied via imbibition or coating, while aiding in waste valorization within the circular economy.

Baiying Juhua Decoction (BYJHD) is a well-established traditional Chinese herbal formula primarily composed of Solanum lyratum and chrysanthemum, which necessitates a thorough investigation to clarify its mechanisms in combating non-small cell lung cancer (NSCLC). This study employed a combination of network pharmacology predictions, serum pharmacochemistry analysis, and various machine learning algorithms (including LASSO, SVM-RFE, and RF) to identify 38 bioactive compounds that target 653 proteins associated with NSCLC. A cross-analysis of 2161 differentially expressed genes (DEGs) and 3124 functional modules led to the identification of 54 critical therapeutic targets. Following this, protein-protein interaction (PPI) and machine learning analysis pinpointed five key signaling regulators. Molecular docking studies demonstrated strong binding affinities between four representative compounds from BYJHD and these targets. Both in vitro and in vivo experiments confirmed that BYJHD inhibits the progression of NSCLC by exerting anti-angiogenic effects, specifically through the inhibition of the ACVRL-1/Smad/ID-1 signaling pathway and the downregulation of CD34. These findings effectively connect traditional clinical applications with contemporary mechanistic insights, positioning BYJHD as a promising multi-target therapeutic candidate for NSCLC.