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Murine adenosine deaminase (mADA) is a prototypic system for studying the thermal activation of active site chemistry within the TIM barrel family of enzyme reactions. Previous temperature-dependent hydrogen deuterium exchange studies under various conditions have identified interconnected thermal networks for heat transfer from opposing protein-solvent interfaces to active site residues in mADA. One of these interfaces contains a solvent exposed helix-loop-helix moiety that presents the hydrophobic face of its long α-helix to the backside of bound substrate. Herein we pursue the time and temperature dependence of solvation dynamics at the surface of mADA, for comparison to established kinetic parameters that represent active site chemistry. We first created a modified protein devoid of native tryptophans with close to native kinetic behavior. Single site-specific tryptophan mutants were back inserted into each of the four positions where native tryptophans reside. Measurements of nanosecond fluorescence relaxation lifetimes and Stokes shift decays, that reflect time dependent environmental reoroganization around the photo-excited state of Trp*, display minimal temperature dependences. These regions serve as controls for the behavior of a new single tryptophan inserted into a solvent exposed region near the helix-loop-helix moiety located behind the bound substrate, Lys54Trp. This installed Trp displays a significantly elevated value for Ea(kStokesshift) ; further, when Phe61 within the long helix positioned behind bound substrate is replaced by a series of aliphatic hydrophobic side chains, the trends in Ea(kStokesshift) mirror the earlier reported impact of the same series of function-altering hydrophobic side chains on the activation energy of catalysis, Ea(kcat) .The reported experimental findings implicate a solvent initiated and rapid (>ns) protein restructuring that contributes to the enthalpic activation barrier to catalysis in mADA.

The analysis of hydrogen deuterium exchange by mass spectrometry as a function of temperature and mutation has emerged as a generic and efficient tool for the spatial resolution of protein networks that are proposed to function in the thermal activation of catalysis. In this work, we extend temperature-dependent hydrogen deuterium exchange from apo-enzyme structures to protein-ligand complexes. Using adenosine deaminase as a prototype, we compared the impacts of a substrate analog (1-deaza-adenosine) and a very tight-binding inhibitor/transition state analog (pentostatin) at single and multiple temperatures. At a single temperature, we observed different hydrogen deuterium exchange-mass spectrometry properties for the two ligands, as expected from their 106-fold differences in strength of binding. By contrast, analogous patterns for temperature-dependent hydrogen deuterium exchange mass spectrometry emerge in the presence of both 1-deaza-adenosine and pentostatin, indicating similar impacts of either ligand on the enthalpic barriers for local protein unfolding. We extended temperature-dependent hydrogen deuterium exchange to a function-altering mutant of adenosine deaminase in the presence of pentostatin and revealed a protein thermal network that is highly similar to that previously reported for the apo-enzyme (Gao et al., 2020, JACS 142, 19936-19949). Finally, we discuss the differential impacts of pentostatin binding on overall protein flexibility versus site-specific thermal transfer pathways in the context of models for substrate-induced changes to a distributed protein conformational landscape that act in synergy with embedded protein thermal networks to achieve efficient catalysis.

Proteins are intrinsically flexible macromolecules that undergo internal motions with time scales spanning femtoseconds to milliseconds. These fluctuations are implicated in the optimization of reaction barriers for enzyme catalyzed reactions. Time, temperature, and mutation dependent hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been previously employed to identify spatially resolved, catalysis-linked dynamical regions of enzymes. We now extend this technique to pursue the correlation of protein flexibility and chemical reactivity within the diverse and widespread TIM barrel proteins, targeting murine adenosine deaminase (mADA) that catalyzes the irreversible deamination of adenosine to inosine and ammonia. Following a structure-function analysis of rate and activation energy for a series of mutations at a second sphere phenylalanine positioned in proximity to the bound substrate, the catalytically impaired Phe61Ala with an elevated activation energy (Ea = 7.5 kcal/mol) and the wild type (WT) mADA (Ea = 5.0 kcal/mol) were selected for HDX-MS experiments. The rate constants and activation energies of HDX for peptide segments are quantified and used to assess mutation-dependent changes in local and distal motions. Analyses reveal that approximately 50% of the protein sequence of Phe61Ala displays significant changes in the temperature dependence of HDX behaviors, with the dominant change being an increase in protein flexibility. Utilizing Phe61Ile, which displays the same activation energy for kcat as WT, as a control, we were able to further refine the HDX analysis, highlighting the regions of mADA that are altered in a functionally relevant manner. A map is constructed that illustrates the regions of protein that are proposed to be essential for the thermal optimization of active site configurations that dominate reaction barrier crossings in the native enzyme.

Objective: To study the false memory among senile normal people with high neuroticism and low neuroticism using neuropsychological scales and event-related potentials (ERPs), and to explore the effects of high neuroticism on false memory and its neuroelectrophysiological mechanism. Methods: A cross-sectional study was conducted, in which the general situation questionnaire, adult version of Eysenck personality questionnaire (EPQ) and Montreal cognitive assessment (MoCA) scale were used to establish a multi-dimensional survey in senile normal people over 60 years old from communities in Zhengzhou, and the EPQ and general situation questionnaire were used to comprehensively screen and divide the study subjects into high neuroticism group and low neuroticism group from 206 senile people. The population was matched by 1:1 according to gender, age (±2 years), and years of education (±2 years), and 40 subjects were finally enrolled for detection of electroencephalograph (EEG) components using ERPs. The Deese-Roediger-McDermott (DRM) paradigm of false memory was designed using E-prime2.0 system, and the stimulus program was presented. The EEG signals of the study subjects were recorded in real time and acquired using 64-channel Neuroscan EEG signals acquisition system. Results: High neuroticism group was evidently lower in the mean accuracy than low neuroticism group, and the difference in the mean accuracy was statistically significant (P = 0.013), but the difference in reaction time was not statistically significant. 2. The mean amplitude of EEG component N400: The difference in the main effect of N400 in the brain region was significantly different (P<0.001), and the mean amplitude of N400 was the largest in frontal region, followed by central region and parietal region successively (all P<0.05). There was no statistically significant difference in the neurotic main effect or the interaction effect of neuroticism and brain region. The latency of N400: There was no significant difference in the neurotic main effect, main effect of the brain region or the interaction effect of neuroticism and brain region. 3. The mean amplitude of EEG component LPC: The difference in the main effect of the brain region was significantly different (P<0.001), and the mean amplitude of LPC was the largest in frontal region, followed by central region and parietal region successively (all P<0.05). There was no significant difference in the neurotic main effect, neuroticism or the interaction effect of neuroticism and brain region. As to the latency of LPC, there was significant difference in the main effect of the brain region (P = 0.025), and the latency of LPC was shorter in frontal region than that in central region (P<0.05). The differences in the neurotic main effect, interaction effect of neuroticism and brain region were not statistically significant. Conclusions: High neuroticism can significantly increase the false memory of senile normal people. The EEG components N400 and LPC are potential early indicators of high neuroticism affecting false memory. High neuroticism may influence false memory by affecting the frontal cortex function. [ABSTRACT FROM AUTHOR]

Simple Summary: Following weaning, piglets exhibit an immature immune system, reduced capacity to digest and absorb nutrients, and encounter diverse stressors that significantly compromise their overall health. The supplementation of probiotics in animal feed has proven to be a beneficial strategy for enhancing the welfare of livestock and poultry. The findings of this research demonstrate that supplementing the diet with 5 × 108 CFU/kg of Clostridium butyricum enhances fecal microflora diversity and richness through the modulation of specific metabolites such as L-aspartic acid, 5-hydroxyindole-3-acetic acid, stearic acid, and adipic acid. This mechanism contributes to a reduction in the incidence of diarrhea in weaned piglets triggered by E. coli K88. Consequently, the study outcomes offer both empirical evidence and theoretical support for the utilization of Clostridium butyricum in combatting diarrhea in piglet rearing. This study aimed to evaluate the effects of Clostridium butyricum (C. butyricum) on the prevention of the diarrhea rates and growth performances of weaned piglets induced by Escherichia coli K88 (E. coli K88). Twenty-four weaned piglets (6.92 ± 0.11 kg) were randomly assigned to one of three treatment groups for a period of 21 days. Each group consisted of eight pigs, with each pig being housed in an individual pen. Group I received the control diet along with normal saline, Group II received the control diet along with E. coli K88, and Group III received the control diet supplemented with 5 × 108 CFU/kg of C. butyricum and E. coli K88. We examined alterations in rectal microbiota and metabolites, analyzed the incidence of diarrhea, and investigated the interactions between microbiota and metabolites through the application of Illumina MiSeq sequencing and liquid chromatography–mass spectrometry. The results showed that, from days 14 to 21, the diarrhea incidence in Group III decreased significantly by 83.29% compared to Group II (p < 0.05). Over the entire experimental duration, the average daily feed intake of Group III decreased significantly by 11.13% compared to Group I (p < 0.05), while the diarrhea incidence in Group III decreased by 71.46% compared to Group II (p < 0.05). The predominant microbial flora in the rectum consisted of Firmicutes (57.32%), Bacteroidetes (41.03%), and Proteobacteria (0.66%). Administering E. coli K88 orally can elevate the relative abundance of Megasphaera (p < 0.05). Conversely, the supplementation of C. butyricum in the diet reduced the relative abundance of Megasphaera (p < 0.05), while increasing the relative abundance of unclassified_f_Lachnospiraceae (p < 0.05). Rectal metabolomics analysis revealed that supplementing C. butyricum in the feed significantly altered the amino acids and fatty acids of the piglets infected with E. coli K88 (p < 0.05). The correlation analysis showed that the occurrence of diarrhea was inversely related to adipic acid (p < 0.05) and positively associated with (5-hydroxyindol-3-YL) acetic acid and L-aspartic acid (p < 0.05). Prevotella_1 exhibited a negative correlation with octadecanoic acid (p < 0.05). Prevotellaceae_UCG-005 showed a negative correlation with (5-hydroxyindol-3-YL) acetic acid (p < 0.05). The findings from this research study aid in probiotic development and the enhancement of healthy growth in weaned piglets. [ABSTRACT FROM AUTHOR]

Background: Individuals suffering from chronic kidney disease (CKD) frequently face a heightened likelihood of experiencing cardiovascular complications, including heart failure and cardiac mortality. Cardiovascular magnetic resonance feature tracking (CMR-FT) is utilized to assess the micro-contraction function of the myocardium. The objective of this research is to explore the relationship between the left ventricular anatomy, myocardial strain, and the clinical outcomes in patients with CKD. Methods: A total of 77 patients with late-stage CKD were enrolled in this retrospective study. They underwent cardiac magnetic resonance imaging and were followed up, with no history of significant cardiac diseases. The patients were divided into two groups: those with a left ventricular global longitudinal strain (LVGLS) ≥ −15.2% (n = 49) and those with LVGLS < −15.2% (n = 28). The clinical endpoints were defined as hospitalization for heart failure or all-cause mortality. Results: Over an average observation period of 22 ± 9 months, 11 (14%) patients passed away and 30 (39%) were admitted to the hospital for heart failure, with eight encountering both incidents. Those with LVGLS ≥ −15.2% had markedly lower rates of event-free survival concerning heart failure admissions and overall mortality than their counterparts (log-rank P = 0.014). Cox multivariable analysis indicated that reduced LVGLS consistently predicted a higher likelihood of combined outcomes of heart failure admissions and total mortality (HR: 3.40, 95% CI [1.35–8.56], P = 0.009), even when factoring in age, diabetes, left atrial diameter, and left ventricular mass index (LVMI). However, the LVMI showed no significant correlation with the risk of heart failure admissions or overall mortality. Conclusion: Compared to patients with LVGLS < −15.2%, CKD patients with LVGLS ≥ −15.2% have an increased risk of heart failure hospitalization and all-cause mortality. The prognostic role of LVMI in assessing CKD patients among the Asian population requires further investigation. [ABSTRACT FROM AUTHOR]