iM-Seeker: a webserver for DNA i-motifs prediction and scoring via automated machine learning.

DNA, beyond its canonical B-form double helix, adopts various alternative conformations, among which the i-motif, emerging in cytosine-rich sequences under acidic conditions, holds significant biological implications in transcription modulation and telomere biology. Despite recognizing the crucial role of i-motifs, predictive software for i-motif forming sequences has been limited. Addressing this gap, we introduce 'iM-Seeker', an innovative computational platform designed for the prediction and evaluation of i-motifs. iM-Seeker exhibits the capability to identify potential i-motifs within DNA segments or entire genomes, calculating stability scores for each predicted i-motif based on parameters such as the cytosine tracts number, loop lengths, and sequence composition. Furthermore, the webserver leverages automated machine learning (AutoML) to effortlessly fine-tune the optimal i-motif scoring model, incorporating user-supplied experimental data and customised features. As an advanced, versatile approach, 'iM-Seeker' promises to advance genomic research, highlighting the potential of i-motifs in cell biology and therapeutic applications. The webserver is freely available at https://im-seeker.org.

G4Atlas: a comprehensive transcriptome-wide G-quadruplex database.

RNA G-quadruplex (rG4) is a vital RNA tertiary structure motif that involves the base pairs on both Hoogsteen and Watson-Crick faces of guanines. rG4 is of great importance in the post-transcriptional regulation of gene expression. Experimental technologies have advanced to identify in vitro and in vivo rG4s across diverse transcriptomes. Building on these recent advances, here we present G4Atlas, the first transcriptome-wide G-quadruplex database, in which we have collated, classified, and visualized transcriptome rG4 experimental data, generated from rG4-seq, chemical profiling and ligand-binding methods. Our comprehensive database includes transcriptome-wide rG4s generated from 82 experimental treatments and 238 samples across ten species. In addition, we have also included RNA secondary structure prediction information across both experimentally identified and unidentified rG4s to enable users to display any potential competitive folding between rG4 and RNA secondary structures. As such, G4Atlas will enable users to explore the general functions of rG4s in diverse biological processes. In addition, G4Atlas lays the foundation for further data-driven deep learning algorithms to examine rG4 structural features.

New insights into thermo-acidophilic properties of Alicyclobacillus acidoterrestris after acid adaptation.

Alicyclobacillus acidoterrestris has unique thermo-acidophilic properties and is the main cause of fruit juice deterioration. Given the acidic environment and thermal treatment during juice processing, the effects of acid adaptation (pH 3.5, 3.2, and 3.0) on the resistance of A. acidoterrestris to heat (65 °C, 5 min) and acid (pH = 2.2, 1 h) stresses were investigated for the first time. The results showed that acid adaptation induced cross-protection against heat stress of A. acidoterrestris and acid tolerance response, and the extent of induced tolerance was increased with the decrease of adaptive pH values. Acid adaptation treatments did not disrupt the membrane potential stability and intracellular pH homeostasis, but reduced intracellular ATP concentration, increased cyclic fatty acids content, and changed the acquired Fourier transform infrared spectra. Transcription levels of stress-inducible (dnaK, grpE, clpP, ctsR) genes and genes related to spore formation (spo0A, ctoX) were up-regulated after acid adaptation, and spore formation was observed by scanning electron microscopy. This study revealed that the intracellular microenvironment homeostasis, expression of chaperones and proteases, and spore formation played a coordinated role in acid stress adaptive responses, with implications for applications in fruit juice processing.

Identification and Characterization of the Small Heat Shock Protein Hsp20 from Oenococcus oeni SD-2a.

Oenococcus oeni can exert its function in hostile wine conditions during the malolactic fermentation process. Therefore, it is an important microbial resource for exploring resistance genes. Hsp20 is an important small heat shock protein from O. oeni. The conserved consensus motif "A-x-x-x-x-G-x-L" of Hsp20 announced its role as a member of the small heat shock protein family. The hsp20 gene from O. oeni SD-2a was cloned to create the recombinant plasmid pTriEx-Hsp20. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and the Hsp20 protein was induced by isopropyl-ß-D-thiogalactoside (IPTG). The hsp20 gene from O. oeni SD-2a was successfully expressed, and a 20-kDa fusion protein was identified by SDS-PAGE. The purified Hsp20 protein was obtained using Ni-affinity chromatography. Additionally, BL21(DE3)/Hsp20 and BL21(DE3)/Ctrl were treated at high temperatures of 42 and 52 °C, at pH values of 2.0-12.0, under oxidative shock with 0.1% (v/v) and 0.2% (v/v) H2O2, and under an osmotic shock of 430 and 860 mM NaCl to compare the effects of heterologous expression of the Hsp20 protein from O. oeni SD-2a for stress resistance. Notably, Hsp20 overexpression showed enhanced resistance than the control strain did when confronted with different elevated stress conditions. The results demonstrated heterologous expression of the hsp20 gene from O. oeni SD-2a significantly improved the resistance of the host E. coli bacteria against stress conditions.

First Insight into the Probiotic Properties of Ten Streptococcus thermophilus Strains Based on In Vitro Conditions.

The aim of this study was to evaluate probiotic properties of ten Streptococcus thermophilus strains (st1 to st10) isolated from pickles in China. These strains all had ß-galactosidase activity, which laid foundation for studying their probiotic properties. In this study, the bile salt hydrolase activity, lysozyme resistance, tolerance to simulated gastric juice, bile salt tolerance, and bacterial adhesion capacity to the Caco-2 cells of these selected strains were detected in vitro conditions. The results indicated that the bile salt hydrolase activities of st2, st6, and st9 were higher than that for other strains. St10 showed the greatest lysozyme resistance (> 80% survival), followed by st9, st8, st7, st5, and st6. As for the tolerance to simulated gastric juice, st5 possessed the highest survival rate (35%), followed by st6 (30%). St6 was the best performer in both bile salt tolerance and bacterial adhesion capacity to the Caco-2 cells. The results of fluorescence microscope and electron microscope further confirmed previous studies and more intuitively demonstrated the st6 strain's tolerance to harsh environments. Overall, these strains were expected to possess beneficial properties and have the potentiality to be probiotics.

Antibacterial activity of selenium-enriched lactic acid bacteria against common food-borne pathogens in vitro.

Selenium (Se) is an essential trace element for human health and animal nutrition. The aim of this study was to evaluate the inhibitory activities of Se-enriched lactic acid bacteria (LAB), Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus, against pathogenic Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes in vitro. The results indicated that the accumulation amount of Se by Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus reached 12.05 ± 0.43 µg/mL and 11.56 ± 0.25 µg/mL, respectively, accompanied by the relative maximum living cells when sodium selenite was 80 µg/mL. Oxford cup double plate assay showed that bacterial culture solution and cell-free culture supernatant (CFCS) from Se-enriched LAB exerted stronger antibacterial activity than those from the non-Se strains. The growth of pathogenic bacterial culture with CFCS at any growth stages was worse than that without CFCS; moreover, the inhibiting effect of CFCS of Se-enriched LAB was more significant than that of non-Se strains. Results from a scanning electron microscope equipped with energy dispersion X-ray spectrometry showed that elemental Se nanoparticles, which characteristically energy peak around 1.42 keV, were deposited on the cell surfaces of Lactobacillus delbrueckii ssp. bulgaricus. In addition, CFCS of Se-enriched LAB induced more serious cell structure damage of pathogenic bacteria than did non-Se LAB.

Deep Learning in RNA Structure Studies.

Deep learning, or artificial neural networks, is a type of machine learning algorithm that can decipher underlying relationships from large volumes of data and has been successfully applied to solve structural biology questions, such as RNA structure. RNA can fold into complex RNA structures by forming hydrogen bonds, thereby playing an essential role in biological processes. While experimental effort has enabled resolving RNA structure at the genome-wide scale, deep learning has been more recently introduced for studying RNA structure and its functionality. Here, we discuss successful applications of deep learning to solve RNA problems, including predictions of RNA structures, non-canonical G-quadruplex, RNA-protein interactions and RNA switches. Following these cases, we give a general guide to deep learning for solving RNA structure problems.

Integrated transcriptomic and proteomic analysis reveals the response mechanisms of Alicyclobacillus acidoterrestris to heat stress.

Alicyclobacillus acidoterrestris can survive pasteurization and is implicated in pasteurized fruit juice spoilage. However, the mechanisms underlying heat responses remain largely unknown. Herein, gene transcription changes of A. acidoterrestris under heat stress were detected by transcriptome, and an integrated analysis with proteomic and physiological data was conducted. A total of 911 differentially expressed genes (DEGs) was observed. The majority of DEGs and differentially expressed proteins (DEPs) were exclusively regulated at the mRNA and protein level, respectively, whereas only 59 genes were regulated at both levels and had the same change trends. Comparative analysis of the functions of the specifically or commonly regulated DEGs and DEPs revealed that the heat resistance of A. acidoterrestris was primarily based on modulating peptidoglycan and fatty acid composition to maintain cell envelope integrity. Low energy consumption strategies were established with attenuated glycolysis, decreased ribosome de novo synthesis, and activated ribosome hibernation. Terminal oxidases, cytochrome bd and aa3, in aerobic respiratory chain were upregulated. Meanwhile, the MarR family transcriptional regulator was upregulated, reactive oxygen species (ROS) was discovered, and the concentration of superoxide dismutase (SOD) increased, indicating that the accompanied oxidative stress was induced by high temperature. Additionally, DNA and protein damage repair systems were activated. This study provided a global perspective on the response mechanisms of A. acidoterrestris to heat stress, with implications for better detection and control of its contamination in fruit juice.

Exploring the catalytic mechanism of a novel ß-glucosidase BGL0224 from Oenococcus oeni SD-2a: Kinetics, spectroscopic and molecular simulation.

The ß-glucosidase derived from microorganisms has attracted worldwide interest for their industrial applications, but studies on ß-glucosidases from Oenococcus oeni are rare. In this paper, catalytic mechanism of a novel ß-glucosidase BGL0224 of Oenococcus oeni SD-2a was explored for the first time by kinetic parameters determination, fluorescence spectroscopy and quenching mechanism analysis, molecular dynamics simulation. The results indicated that BGL0224 had universal catalytic effect on different types of glycoside substrates, but the catalytic efficiencies were different. Fluorescence quenching analysis results suggested that the quenching processes between BGL0224 and seven kinds of substrates were predominated by the static quenching mechanism. A reasonable three-dimensional model of BGL0224 was obtained using the crystal structure of E.coli BglA as a template. The analysis results of molecular simulation (RMSD, Rg, RMSF and hydrogen bonding) showed that the composite system 'BGL0224-pNPG' was very stable after 40 ns. The catalytic process of BGL0224 acting on 'p-Nitrophenyl ß-d-glucopyranoside' conformed to the double displacement mechanism. Two glutamic acid residues 'Glu178 and Glu377' played a vital role in the whole catalytic process. Overall, this study gave specific insights on the catalytic mechanism of BGL0224, which was of great significance for developing its potential applications in food industry.

Altered Metabolic Strategies: Elaborate Mechanisms Adopted by Oenococcus oeni in Response to Acid Stress.

Oenococcus oeni plays a key role in inducing malolactic fermentation in wine. Acid stress is often encountered under wine conditions. However, the lack of systematic studies of acid resistance mechanisms limits the downstream fermentation applications. In this study, the acid responses of O. oeni were investigated by combining transcriptome, metabolome, and genome-scale metabolic modeling approaches. Metabolite profiling highlighted the decreased abundance of nucleotides under acid stress. The gene-metabolite bipartite network showed negative correlations between nucleotides and genes involved in ribosome assembly, translation, and post-translational processes, suggesting that stringent response could be activated under acid stress. Genome-scale metabolic modeling revealed marked flux rerouting, including reallocation of pyruvate, attenuation of glycolysis, utilization of carbon sources other than glucose, and enhancement of nucleotide salvage and the arginine deiminase pathway. This study provided novel insights into the acid responses of O. oeni, which will be useful for designing strategies to address acid stress in wine malolactic fermentation.

Homology analysis of 35 ß-glucosidases in Oenococcus oeni and biochemical characterization of a novel ß-glucosidase BGL0224.

ß-Glucosidases play an important role in food industry. Oenococcus oeni are typical lactic acid bacteria that initiate malolactic fermentation of wines. 35 ß-glucosidases from O. oeni were selected and their conserved domains and evolutionary relationships were further explored in this study. The homology analysis results indicated that 35 ß-glucosidases were basically derived from GH1 and GH3 family. A novel ß-glucosidase was successfully expressed and characterized. The recombinant protein, referred to as BGL0224, consisted of a total 480 amino acids with an apparent molecular weight of 55.15 kDa and was classified as GH1 family. It achieved the highest activity at pH 5.0 and 50 °C. The activity and stability were significantly increased when 12% ethanol was supplemented to the enzyme. Using p-NPG as substrate, the Km, Vmax and Kcat of BGL0224 were 0.34 mM, 382.81 U/mg and 351.88 s-1, respectively. In all, BGL0224 has good application prospects in food industry.

Multivariate analysis reveals effect of glutathione-enriched inactive dry yeast on amino acids and volatile components of kiwi wine.

Principal component analysis (PCA) and partial least squares (PLS) regression were applied to investigate the effect of glutathione-enriched inactive dry yeast (g-IDY) on the amino acids and volatile components of kiwi wine. Results indicated that the addition of g-IDY had positive effect on most amino acids of kiwi wine, especially glutamine and glycine. In case of pure juice fermentation, the concentrations of ethyl decanoate, 2-methylbutyric acid, trans-2-nonenal and hexyl butyrate had notably positive correlation with the addition of g-IDY. PLS regression indicated that the amino acids were highly interrelated to the volatile compositions, and glycine had the strongest positive impact on the concentrations of esters and total volatile components. This might explain the similar effect of g-IDY on the amino acids and volatile components of kiwi wine. Besides, PLS regression showed that E-nose was a good method to predict volatile compositions of kiwi wine, especially esters.

Effect of reduced glutathione on the quality characteristics of apple wine during alcoholic fermentation.

To investigate the effect of reduced glutathione (GSH) on the quality characteristics of apple wine, 10, 20 and 30 mg/L of GSH were added to apple juice before alcoholic fermentation. Meanwhile, apple wine fermented by Saccharomyces cerevisiae which had been pre-incubated with GSH (100 mg/L) was another experimental group. Mono-phenols, GSH and oxidized glutathione (GSSG) were determined by HPLC. Aroma compounds were analysed by GC-MS. Further, E-nose was applied to monitor the odor. After fermentation, GSH content was the same in all of the samples. However, for the apple wine with GSH addition, GSSG content increased significantly. Notably, GSH could reduce the color index, protect chlorogenic acid and phloretin, decrease the content of epicatechin and catechin as well as change the profile of aroma compounds (higher levels of 2-methyl-1-propanol, 3-methyl-1-butanol, ethyl benzoate, linalool, etc.). GSH may be used for flavor enhancement and quality improvement of apple wine.

Analysis of proteomic responses of freeze-dried Oenococcus oeni to access the molecular mechanism of acid acclimation on cell freeze-drying resistance.

Malolactic fermentation (MLF), usually induced by Oenococcus oeni (O. oeni), is an important process to improve wine quality. Acid acclimation has been proven to be useful for enhancing the viability of lyophilized O. oeni. To explain the involved mechanisms, cell integrity, morphology and protein patterns of lyophilized O. oeni SD-2a were investigated with acid acclimation. After lyophilization, improvement of cell integrity and more extracellular polymeric substances (EPS) were observed in acid acclimated cells. Combined with GO and KEGG analysis, different abundant proteins were noticeably enriched in the carbohydrate metabolism process, especially amino sugar and nucleotide sugar metabolism. The most significant result was the over-expression of proteins participating in cell wall biosynthesis, EPS production, ATP binding and the bacterial secretion system. This result indicated the important role of acid acclimation on cell envelope properties. In addition, protein response to stress and arginine deiminase pathway were also proven to be over-expressed.

Quantitative proteomics analysis reveals proteins and pathways associated with anthocyanin accumulation in barley.

The aim of the present study was to explore the underlying mechanisms involved in anthocyanin biosynthesis in purple, blue, and white barley using quantitative proteomics analysis. We identified the differences in protein expression and related functions involved in anthocyanin biosynthesis in purple, blue, and white barley (named H, M, and L groups, respectively, based on their anthocyanin content) using TMT-liquid chromatography/mass spectroscopy-based proteomic methods. Totally, 297, 300, 254, and 1421 differentially expressed proteins (DEPs) were found in H vs. L, H vs. M, L vs. M, and H vs. L vs. M groups, respectively. Six clusters of proteins from the 1421 DEPs were mainly involved in carbon metabolism, amino acid and secondary metabolite biosynthesis, and metabolic pathways. Several proteins were validated using parallel reaction monitoring. The proteins involved in amino acid biosynthesis, carbon metabolism, metabolic pathways, and phenylpropanoid biosynthesis were responsible for the color differences in the three barley varieties.

Surface characteristics and proteomic analysis insights on the response of Oenococcus oeni SD-2a to freeze-drying stress.

Oenococcus oeni (O. oeni) induces malolactic fermentation to improve wine quality. However, the molecular basis of mechanisms involved in its freeze-drying resistance remains unclear. In this work, O. oeni SD-2a without freeze-drying (No-FD), with freeze-drying (FD) and freeze-drying with monosodium glutamate (MSG) (FD-MSG) were investigated. Flow cytometry results showed severe cell damage in FD cells and less membrane transmissibility in FD-MSG cells. Meanwhile, extracellular polymeric substances (EPS) were detected in FD and FD-MSG cells by scanning electron microscopy. Bioinformatic analysis revealed that varying proteins were involved in carbon, lipid and nucleic acids metabolism, stress response, oxidoreductase activity and signal sensing. Among the identified proteins, the highlighted proteins were those involved in polysaccharides production and signal sensing for freeze-drying resistance and cyclopropane fatty acid metabolism for MSG addition.