Isothermal Titration Calorimetry Research Articles

Assessment of the Interaction of Acetylcholinesterase Binding with Bioactive Compounds from Coffee and Coffee Fractions Digested In Vitro in the Gastrointestinal Tract.

The aim of the study was to evaluate the degree of acetylcholinesterase (AChE) inhibition by green and light- and dark-roasted coffee extracts and their fractions after digestion in a simulated gastrointestinal tract. The analysis was carried out using isothermal titration calorimetry, molecular docking, and dynamics simulations. The results showed that 3-O-caffeoylquinic acid binds strongly to AChE through hydrogen interactions with the amino acids ARG289A, HIS440A, and PHE288A and hydrophobic interactions with TYR121A in the active site of the enzyme. The Robusta green coffee extract (ΔG = -35.87 kJ/mol) and dichlorogenic acid fraction (ΔG = -19-29 kJ/mol) showed the highest affinity. Dichlorogenic acids (3,4-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, and 3,4-O-dicaffeoylquinic acid) have high affinity for AChE as single compounds (ΔG(ITC) = -48.99-55.36 kJ/mol, ΔG(LF/AD) = -43.38-45.38 kJ/mol). The concentration necessary to reduce AChE activity by 50% amounted to 0.22 μmol/μmol chlorogenic acids to the enzyme.

Solvation-Enhanced Salt Bridges.

Salt bridges formed by amidines and carboxylic acids represent an important class of noncovalent interaction in biomolecular and supramolecular systems. Isothermal titration calorimetry was used to study the relationships between the strength of the interaction, the chemical structures of the components, and the nature of the solvent. The stability of the 1:1 complex formed in chloroform changed by 2 orders of magnitude depending on the basicity of the amidine and the acidity of the acid, which is consistent with proton transfer in the complex. Polar solvents reduce the stabilities of salt bridges formed with N,N'-dialkylamidines by up to 3 orders of magnitude, but this dependence on solvent polarity can be eliminated if the alkyl groups are replaced by protons in the parent amidine. The enhanced stability of the complex formed by benzamidine is due to solvation of the NH sites not directly involved in salt bridge formation, which become significantly more polar when proton transfer takes place, leading to more favorable interactions with polar solvents in the bound state. Calculation of H-bond parameters using density functional theory was used to predict solvent effects on the stabilities of salt bridges to within 1 kJ mol-1. While H-bonding interactions are strong in nonpolar solvents, and solvophobic interactions are strong in polar protic solvents, these interactions are weak in polar aprotic solvents. In contrast, amidinium-carboxylate salt bridges are stable in both polar and nonpolar aprotic solvents, which is attractive for the design of supramolecular systems that operate in different solvent environments.

PHD-BAH Domain in ASH1L Could Recognize H3K4 Methylation and Regulate the Malignant Behavior of Cholangio Carcinoma.

Histone methyltransferase absent, small, or homeotic discs1-like (ASH1L) is composed of su(var)3-9, enhancer of zeste, trithorax (SET) domain, pleckstrin homology domain (PHD) domain, middle (MID) domain, and bromo adjacent homology (BAH) domain. The SET domain of ASH1L is known to mediate mediate H3K36 dimethylation (H3K36me2) modification. However, the specific functions of the PHD-BAH domain remain largely unexplored. This study aimed to explore the biological function of the PHD-BAH domain in ASH1L. We employed a range of techniques, including a prokaryotic fusion protein expression purification system, pull-down assay, Isothermal Titration Calorimetry (ITC), polymerase chain reaction (PCR), and sitedirected mutagenesis, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) gene editing, cell culture experiment, western blot, cell proliferation assay, and cell apoptosis test. The PHD-BAH domain in ASH1L preferentially binds to the H3K4me2 peptide over H3K4 monomethylation (H3K4me1) and H3K4 trimethylation (H3K4me3) peptide. Notably, the W2603A mutation within the PHD-BAH domain could disrupt the interaction with H3K4me2 in vitro. Compared with wild-type Cholangiocarcinoma (CHOL) cells, deletion of the PHD-BAH domain in ASH1L led to increased CHOL cell apoptosis and reduced cell proliferation (P < 0.001). Additionally, the W2603A mutation affected the regulation of the proteasome 20S subunit beta (PSMB) family gene set. W2603A mutation was crucial for the interaction between the PHD-BAH domain and the H3K4me2 peptide. ASH1L regulated CHOL cell survival and proliferation through its PHD-BAH domain by modulating the expression of the PSMB family gene set.

Fabrication of a Heptapeptide-Modified Poly(glycidyl Methac-Rylate) Nanosphere for Oriented Antibody Immobilization and Immunoassay

Oriented antibody immobilization has been widely employed in immunoassays and immunodiagnoses due to its efficacy in identifying target antigens. Herein, a heptapeptide ligand, HWRGWVC (HC7), was coupled to poly(glycidyl methacrylate) (PGMA) nanospheres (PGMA-HC7). The antibody immobilization behavior and antigen recognition performance were investigated and compared with those on PGMA nanospheres by nonspecific adsorption and covalent coupling via carbodiimide chemistry. The antibodies tested included bovine, rabbit, and human immunoglobulin G (IgG), while the antigens included horseradish peroxidase (HRP) and β-2-Microglobulin (β2-MG). The nanospheres were characterized using zeta potential and particle size analyzers, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and reversed-phase chromatography, proving each synthesis step was succeeded. Isothermal titration calorimetry assay demonstrated the strong affinity interaction between IgG and PGMA-HC7. Notably, PGMA-HC7 achieved rapid and extremely high IgG adsorption capacity (~3 mg/mg) within 5 min via a specific recognition via HC7 without nonspecific interactions. Moreover, the activities of immobilized anti-HRP and anti-β2-MG antibodies obtained via affinity binding were 1.5-fold and 2-fold higher than those of their covalent coupling counterparts. Further, the oriented-immobilized anti-β2-MG antibody on PGMA-HC7 exhibited excellent performance in antigen recognition with a linear detection range of 0–5.3 μg/mL, proving its great potential in immunoassay applications.

Structure-guided mutations in CDRs for enhancing the affinity of neutralizing SARS-CoV-2 nanobody

The optimization of antibodies to attain the desired levels of affinity and specificity holds great promise for the development of next generation therapeutics. This study delves into the refinement and engineering of complementarity-determining regions (CDRs) through in silico affinity maturation followed by binding validation using isothermal titration calorimetry (ITC) and pseudovirus-based neutralization assays. Specifically, it focuses on engineering CDRs targeting the epitopes of receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. A structure-guided virtual library of 112 single mutations in CDRs was generated and screened against RBD to select the potential affinity-enhancing mutations. Protein-protein docking analysis identified 32 single mutants of which nine mutants were selected for molecular dynamics (MD) simulations. Subsequently, biophysical ITC studies provided insights into binding affinity, and consistent with in silico findings, six mutations that demonstrated better binding affinity than native nanobody were further tested in vitro for neutralization activity against SARS-CoV-2 pseudovirus. Leu106Thr mutant was found to be most effective in virus-neutralization with IC50 values of ∼0.03 μM, as compared to the native nanobody (IC50 ∼0.77 μM). Thus, in this study, the developed computational pipeline guided by structure-aided interface profiles and thermodynamic analysis holds promise for the streamlined development of antibody-based therapeutic interventions against emerging variants of SARS-CoV-2 and other infectious pathogens.

The Spatial Distribution of Lipophilic Cations in Gradient Copolymers Regulates Polymer-pDNA Complexation, Polyplex Aggregation, and Intracellular pDNA Delivery.

Here, we demonstrate that the spatial distribution of lipophilic cations governs the complexation pathways, serum stability, and biological performance of polymer-pDNA complexes (polyplexes). Previous research focused on block/statistical copolymers, whereas gradient copolymers, where the density of lipophilic cations diminishes (gradually or steeply) along polymer backbones, remain underexplored. We engineered gradient copolymers that combine the polyplex colloidal stability of block copolymers with the transfection efficiency of statistical copolymers. We synthesized length- and compositionally equivalent gradient copolymers (G1-G3) along with statistical (S) and block (B) copolymers of 2-(diisopropylamino)ethyl methacrylate and 2-hydroxyethyl methacrylate. We mapped how polymer microstructure governs pDNA loading per polyplex, pDNA conformational changes, and polymer-pDNA binding thermodynamics via static light scattering, circular dichroism spectroscopy, and isothermal titration calorimetry, respectively. While gradient steepness is a powerful design handle to improve polyplex physical properties, augment pDNA delivery capacity, and attenuate polycation-triggered hemolysis, microstructural contrasts did not elicit differences in complement activation.

Plant protein-based complex coacervation via protein deamidation

Protein-polysaccharide complex coacervates, as common electrostatic assembly, have been widely used in probiotic encapsulation, emulsifiers, and binders. However, due to their own compact structure, plant proteins usually complex with polysaccharides to form solid-like precipitates via liquid-solid phase separation (LSPS) instead of droplet-like coacervates via liquid-liquid phase separation (LLPS). In this study, we adopted an enzymatic deamidation approach to promote the transition of protein-polysaccharide complexes from precipitates to coacervates. β-conglycinin (β-CG), as a common plant protein, was modified by protein-glutaminase (PG) and complexed with chitosan oligosaccharide (COS) to obtain droplet-like coacervates. Native-PAGE and SDS-PAGE results demonstrated that depolymerization of β-CG occurred during deamidation, leading to the production of oligomers and changes in the surface charge patchiness of β-CG. Isothermal titration calorimetry (ITC) results showed that precipitates were mainly driven by exotherm, while coacervates were driven by entropy gain. This study offers a novel approach for the generation of plant protein-based coacervates.

Activation of parkin by a molecular glue

Mutations in parkin and PINK1 cause early-onset Parkinson’s disease (EOPD). The ubiquitin ligase parkin is recruited to damaged mitochondria and activated by PINK1, a kinase that phosphorylates ubiquitin and the ubiquitin-like domain of parkin. Activated phospho-parkin then ubiquitinates mitochondrial proteins to target the damaged organelle for degradation. Here, we present the mechanism of activation of a new class of small molecule allosteric modulators that enhance parkin activity. The compounds act as molecular glues to enhance the ability of phospho-ubiquitin (pUb) to activate parkin. Ubiquitination assays and isothermal titration calorimetry with the most active compound (BIO-2007817) identify the mechanism of action. We present the crystal structure of a closely related compound (BIO-1975900) bound to a complex of parkin and two pUb molecules. The compound binds next to pUb on RING0 and contacts both proteins. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments confirm that activation occurs through release of the catalytic Rcat domain. In organello and mitophagy assays demonstrate that BIO-2007817 partially rescues the activity of parkin EOPD mutants, R42P and V56E, offering a basis for the design of activators as therapeutics for Parkinson’s disease.

Solvation free energy in governing equations for DNA hybridization, protein-ligand binding, and protein folding.

This work examines the thermodynamics of model biomolecular interactions using a governing equation that accounts for the participation of bulk water in the equilibria. In the first example, the binding affinities of two DNA duplexes, one of nine and one of 10 base pairs in length, are measured and characterized by isothermal titration calorimetry (ITC) as a function of concentration. The results indicate that the change in solvation free energy that accompanies duplex formation (ΔGS) is large and unfavorable. The duplex with the larger number of G:C pairings yields the largest change in solvation free energy, ΔGS = +460 kcal·mol-1per base pair at 25 °C. A van't Hoff analysis of the data is complicated by the varying degree of intramolecular base stacking within each DNA strand as a function of temperature. A modeling study reveals how the solvation free energy alters the output of a typical ITC experiment and leads to a good, though misleading, fit to the classical equilibrium equation. The same thermodynamic framework is applied to a model protein-ligand interaction, the binding of ribonuclease A with the nucleotide inhibitor 3'-UMP, and to a conformational equilibrium, the change in tertiary structure of α-lactalbumin in molar guanidinium chloride solutions. The ribonuclease study yields a value of ΔGS = +160 kcal·mol-1, whereas the folding equilibrium yields ΔGS ≈ 0, an apparent characteristic of hydrophobic interactions. These examples provide insight on the role of solvation energy in binding equilibria and suggest a pivot in the fundamental application of thermodynamics to solution chemistry.

Thermodynamic origin of the affinity, selectivity and domain specificity of metallothionein for essential and toxic metal ions.

The small Cys-rich protein metallothionein (MT) binds several metal ions in clusters within its two domains. While the affinity of MT for both toxic and essential metals has been well studied, the thermodynamics of this binding has not. We have used isothermal titration calorimetry measurements to quantify the change in enthalpy (ΔH) and change in entropy (ΔS) when metal ions bind to the two ubiquitous isoforms of MT. The seven Zn2+ that bind sequentially at pH 7.4 do so in two populations with different coordination thermodynamics, an initial four that bind randomly with individual tetra-thiolate coordination and a subsequent three that bind with bridging thiolate coordination to assemble the metal clusters. The high affinity of MT for both populations is due to a very favourable binding entropy that far outweighs an unfavourable binding enthalpy. This originates from a net enthalpic penalty for Zn2+ displacement of protons from the Cys thiols and a favourable entropic contribution from the displaced protons. The thermodynamics of other metal ions binding to MT were determined by their displacement of Zn2+ from Zn7MT and subtraction of the Zn2+-binding thermodynamics. Toxic Cd2+, Pb2+ and Ag+, and essential Cu+, also bind to MT with a very favourable binding entropy but a net binding enthalpy that becomes increasingly favourable as the metal ion becomes a softer Lewis acid. These thermodynamics are the origin of the high affinity, selectivity and domain specificity of MT for these metal ions and the molecular basis for their in vivo binding competition.

Identification and biophysical characterization of potential phytochemical inhibitors of carboxyl/choline esterase from Helicoverpa armigera for advancing integrated pest management strategies

In the realm of disease vectors and agricultural pest management, insecticides play a crucial role in preserving global health and ensuring food security. The pervasive use, particularly of organophosphates (OPs), has given rise to a substantial challenge in the form of insecticide resistance. Carboxylesterases emerge as key contributors to OP resistance, owing to their ability to sequester or hydrolyze these chemicals. Consequently, carboxylesterase enzymes become attractive targets for the development of novel insecticides. Inhibiting these enzymes holds the potential to restore the efficacy of OPs against which resistance has developed. This study aimed to screen the FooDB library to identify potent inhibitory compounds targeting carboxylesterase, Ha006a from the agricultural pest Helicoverpa armigera. The ultimate objective is to develop effective interventions for pest control. The compounds with the highest scores underwent evaluation through docking studies and pharmacophore analysis. Among them, four phytochemicals—donepezil, protopine, 3′,4′,5,7-tetramethoxyflavone, and piperine—demonstrated favorable binding affinity. The Ha006a-ligand complexes were subsequently validated through molecular dynamics simulations. Biochemical analysis, encompassing determination of IC50 values, complemented by analysis of thermostability through Differential Scanning Calorimetry and interaction kinetics through Isothermal Titration Calorimetry was conducted. This study comprehensively characterizes Ha006a-ligand complexes through bioinformatics, biochemical, and biophysical methods. This investigation highlights 3′,4′,5,7-tetramethoxyflavone as the most effective inhibitor, suggesting its potential for synergistic testing with OPs. Consequently, these inhibitors offer a promising solution to OP resistance and address environmental concerns associated with excessive insecticide usage, enabling a significant reduction in their overuse.

Interaction between reacetylated chitosan and albumin in alcalescent media

Interaction of chitosan and its derivatives with proteins of animal blood at blood pH relevant conditions is of a particular interest for construction of antimicrobial chitosan/protein-based drug delivery systems. In this work, the interaction of a series of N-reacetylated oligochitosans (RA-CHI) having Mw of 10–12 kDa and differing in the degree of acetylation (DA 19, 24, and 40 %) with bovine serum albumin (BSA) in alkalescent media is described in first. It is shown that RA-CHI forms soluble complexes with BSA in solutions with pH 7.4 and a low ionic strength. Light scattering study shows that soluble RA-CHI complexes have spherical form with the radius of about 100 nm. Circular dichroism, fluorescent spectroscopy, and micro-IR spectroscopy studies show that the secondary structure of BSA in soluble complexes remain intact. Isothermal titration calorimetry of RA-CHI with DA 24 % and BSA mixing in the buffers with different ionization heats reveals a significant contribution of electrostatic forces to the binding process and an additional ionization of chitosan due to the proton transfer from the buffer substance. An increase of ionic strength to the blood relevant value 0.15 M suppresses the binding. It is shown that application of RA-CHI with higher DA value leads to a decrease in the affinity of RA-CHI to BSA and an alteration of the interaction mechanism. The finding opens an opportunity to the application of N-reacetylated chitosan derivatives in the complex systems compatible with blood plasma proteins.

Interactions between Hyaluronic Acid and Chitosan by Isothermal Titration Calorimetry: The Effect of Ionic Strength, pH, and Polymer Molecular Weight.

Hyaluronic acid (HA)/chitosan (CHI) complex coacervates have recently gained interest due to the pH-dependent ionization and semiflexibility of the polymers as well as their applicability in tissue engineering. Here, we apply isothermal titration calorimetry (ITC) to understand the apparent thermodynamics of coacervation for HA/CHI as a function of the pH, ionic strength, and chain length. We couple these ITC experiments with the knowledge of the charge states of HA and CHI from potentiometric titration to understand the mechanistic aspects of complex formation. Our data demonstrate that the driving force for the complex coacervation of HA and CHI is entropic in nature and this driving force decreased with increasing ionic strength. We also observed a decrease in the stoichiometry for ion-pairing with increasing ionic strength, which we suggest is a consequence of the changing degree of ionization for HA at higher ionic strengths. An increase in the strength of interactions with pH was hypothesized to also be a result of changes in the degree of ionization of HA, though stronger interactions were observed at the lowest pH tested, likely due to contributions from hydrogen bonding between HA and CHI.

A DNA Aptamer for 2-Aminopurine: Binding-induced Fluorescence Quenching.

2-Aminopurine (2AP) is a fluorescent analog of adenine, and its unique properties make it valuable in various scientific and biotechnological applications. Its fluorescence property probes local dynamics in DNA and RNA because the surrounding bases quench its fluorescence. 2AP-labeled probes that can bind to specific DNA or RNA sequences, enabling the detection of genetic mutations, viral RNA, or other nucleic acid-based markers associated with diseases like cancer and infectious diseases. In this study, we isolated aptamers for 2AP using the library immobilization capture-SELEX technique. Two major aptamer families were isolated after 15 rounds of screening. The Kd values for the 2AP1 aptamer from family 1 are 209 nM in a fluorescence assay and 72 nM in an isothermal titration calorimetry test. The 32 nM 2AP limit of detection was tested. Additionally, we conducted some mutation analysis. Furthermore, we tested the selectivity of our aptamer using various molecules with similar structures and discovered that it can bind adenine and adenosine as well.

Elucidating the Binding Interaction of a Highly Efficient Phytochemical-Sinapic Acid with Human Serum Albumin: A Spectroscopic and Calorimetric Approach

Polyphenolic compounds have received much attention due to their major function as antioxidants. Sinapic acid (SA) is an orally bioavailable phytochemical with excellent free radical scavenging property transported in the blood by human serum albumin (HSA) which is the chief carrier protein found abundantly in human plasma. SA contains a single phenolic group. This study investigates the binding and interaction mechanism between SA and HSA by multiple spectroscopic and calorimetric techniques, such as UV/visible absorption spectroscopy, intrinsic fluorescence studies, Fourier transform infrared (FTIR) spectroscopy and isothermal titration calorimetry (ITC). UV/visible absorption spectroscopy showed hyperchromicity in the absorption spectra, suggestive of structural change due to complex formation between HSA and SA. Intrinsic fluorescence measurements, performed at three different temperatures, showed quenching of the fluorescence spectra and the mechanism of quenching was static in nature which occurred due to ground state complex formation. The FTIR results demonstrated a 16% decrease in α-helical content of the secondary structure of HSA in the presence of SA. Hydrogen bonds and hydrophobic forces were the main forces of interaction, according to the thermodynamic characteristics found through ITC. Moreover, the data obtained through ITC indicated strong binding affinity between HSA and SA and supported the exothermic and spontaneous nature of the interaction.

Biophysical and Solution Structure Analysis of Critical Residues Involved in the Interaction between the PupB N-Terminal Signaling Domain and PupR C-Terminal Cell Surface Signaling Domain from Pseudomonas capeferrum.

Cell surface signaling (CSS) is a means of rapidly adjusting transcription in response to extracellular stimuli in Gram-negative bacteria. The pseudobactin BN7/8 uptake (Pup) system not only imports iron but also upregulates its own transcription through CSS in Pseudomonas capeferrum. In the absence of ferric pseudobactin BN7/8, the signaling components are maintained in a resting state via the formation of a periplasmic complex between the N-terminal signaling domain (NTSD) of the outer membrane iron-transporter, PupB, and the C-terminal CSS domain (CCSSD) of the sigma regulator, PupR. The previously determined 1.6 Å crystal structure of this periplasmic complex has allowed us to probe the structural and thermodynamic consequences of mutating key interfacial residues. In this report, we describe the solution structure of the PupB NTSD and use Nuclear Magnetic Resonance spectroscopy, Isothermal Titration Calorimetry, and Circular Dichroism spectroscopy together with thermal denaturation to investigate whether three PupB point mutations, Q69K, H72D, and L74A, influence the interaction merely due to the chemical nature of the amino acid substitution or also cause changes in overall protein structure. Our results demonstrate that binding to the PupR CCSSD does not alter the structure of PupB NTSD and that the individual mutations have only minor effects on structure. The mutations generally lower thermodynamic stability of the NTSD and weaken binding to the CCSSD. These findings validate the X-ray crystal structure interface, emphasizing the importance of amino acid chemical nature at the interface.

Interaction of an anticancer oxygenated propenylbenzene derivatives with human topoisomerase II α and actin: molecular modeling and isothermal titration calorimetry studies

AbstractCancer diseases are one of the most common causes of death. It is important to reduce the proliferation of cancer cells at an early stage, but also to limit their migration. There is a need to find new compounds of moderate anticancer prevention activity for long administration. TOPIIα and actin are proteins that in states of inflammation can cause the progression of cancer and neoblastic cell migrations. Looking for compounds that will work comprehensively in preventing cancer, interacting with both TOPIIα and actin is crucial/was our aim. In this study, the antioxidant properties of propenylbenzene derivatives and their affinity to bind actin and TOPIIα causing inhibition of their functions were evaluated. The ligand–protein binding assay was carried out by isometric titration calorimetry (ITC), and molecular docking, and the antioxidant potential. The highest chelation activity was shown by 5b: 83.95% (FRAP 18.39 μmol Fe(II) mL−1). High affinity for actin and TOPIIα using ITC and docking was shown by diol forms. For actin the best ligands were 2b (∆H − 51.49 kJ mol−1, ∆G − 27.37 kJ mol−1) and 5b (∆H − 17.25 kJ mol−1, ∆G − 26.20 kJ mol−1), whereas for TOPIIα: 3b (∆H − 163.86 kJ mol−1, ∆G − 34.60 kJ mol−1) and 5b (∆H − 160.93 kJ mol−1, ∆G − 32.92 kJ mol−1). To confirm the occurrence of the interactions at the active site of the proteins, molecular docking and subsequent molecular dynamics simulations were performed, which showed for both actin and TOPIIα the highest enthalpy of interactions of 5b: − 34.94 kJ mol−1 and − 25.52 kJ mol−1, respectively.

Monitoring and predicting the enzymatic inhibition of α‐amylase by apple extracts using Isothermal Titration Calorimetry and Random Forest

Monitoring and predicting the enzymatic inhibition of α‐amylase by apple extracts using Isothermal Titration Calorimetry and Random Forest

The phytopathogen Xanthomonas campestris senses and effluxes salicylic acid via a sensor HepR and an RND family efflux pump to promote virulence in host plants.

Salicylic acid (SA) plays an essential role in plant defense against biotrophic and semi-biotrophic pathogens. Following pathogen recognition, SA biosynthesis dramatically increases at the infection site of the host plant. The manner in which pathogens sense and tolerate the onslaught of SA stress to survive in the plant following infection remains to be understood. The objective of this work was to determine how the model phytopathogen Xanthomonas campestris pv. campestris (Xcc) senses and effluxes SA during infection inside host plants. First, RNA-Seq analysis identified an SA-responsive operon Xcc4167-Xcc4171, encoding a MarR family transcription factor HepR and an RND (resistance-nodulation-cell division) family efflux pump HepABCD in Xcc. Electrophoretic mobility shift assays and DNase I footprint analysis revealed that HepR negatively regulated hepABCD expression by specifically binding to an AT-rich region of the promoter of the hepRABCD operon, Phep. Second, isothermal titration calorimetry and further genetic analysis suggest that HepR is a novel SA sensor. SA binding released HepR from its cognate promoter Phep and then induced the expression of hepABCD. Third, the RND family efflux pump HepABCD was responsible for SA efflux. The hepRABCD cluster was also involved in the regulation of culture pH and quorum sensing signal diffusible signaling factor turnover. Finally, the hepRABCD cluster was transcribed during the XC1 infection of Chinese radish and was required for the full virulence of Xcc in Chinese radish and cabbage. These findings suggest that the ability of Xcc to co-opt the plant defense signal SA to activate the multidrug efflux pump may have evolved to ensure Xcc survival and virulence in susceptible host plants.

The regulatory mechanism of mannan from millet Huangjiu on flavor release

Huangjiu, the national wine of China, has the characteristics of low grain consumption, low alcohol content and high nutrition, which aligns with the world's beverage wine consumption trend. The research on glutinous rice huangjiu from southern China is more extensive, while millet huangjiu from northern China has received less attention, hindering the diversified development of huangjiu. Polysaccharides play an important role in huangjiu's health activity and flavor characteristics. In this study, the polysaccharide components in millet huangjiu were separated and identified for the first time, and the influence of polysaccharides on the flavor release was explored through sensory evaluation, SPME-GC–MS, threshold determination and isothermal titration calorimetry. The millet huangjiu polysaccharide HJ-1 was composed of →3)-α-Manp(1→, α-Manp-(1→, →2,6)-α-Manp-(1→, →3)-α-Xylp-(1→ and →3)-β-Arap-(1→. HJ-1 (0.1–1 mg/mL) could regulate the release performance of key flavor compounds in millet huangjiu, inhibit the herbal, fruity and alcoholic aromas, and promote the sweet and rice aromas. Further studies showed that HJ-1 formed complexes with ethyl acetate, acetic acid, 2,3-butanediol and γ-butyrolactone through van der Waals forces and hydrogen bonds, and the process was a spontaneous exothermic entropy reduction reaction. This study provides a new idea for optimizing the overall aroma of huangjiu and improving its sensory quality.