Impact of Western Diet on Enterohemorrhagic Escherichia coli Colonization in the Human In Vitro Mucosal Artificial Colon as Mediated by Gut Microbiota.

Enterohemorrhagic Escherichia coli (EHEC) is a major food-borne pathogen that causes human disease ranging from diarrhea to life-threatening complications. Accumulating evidence demonstrates that the Western diet enhances the susceptibility to enteric infection in mice, but the effect of diet on EHEC colonization and the role of human gut microbiota remains unknown. Our research aimed to investigate the effects of a Standard versus a Western diet on EHEC colonization in the human in vitro Mucosal ARtificial COLon (M-ARCOL) and the associated changes in the gut microbiota composition and activities. After donor selection using simplified fecal batch experiments, two M-ARCOL bioreactors were inoculated with a human fecal sample (n = 4) and were run in parallel, one receiving a Standard diet, the other a Western diet and infected with EHEC O157:H7 strain EDL933. EHEC colonization was dependent on the donor and diet in the luminal samples, but was maintained in the mucosal compartment without elimination, suggesting a favorable niche for the pathogen, and may act as a reservoir. The Western diet also impacted the bacterial short-chain fatty acid and bile acid profiles, with a possible link between high butyrate concentrations and prolonged EHEC colonization. The work demonstrates the application of a complex in vitro model to provide insights into diet, microbiota, and pathogen interactions in the human gut.

De novo design of proteins housing excitonically coupled chlorophyll special pairs.

Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.

Exploring Metabolic and Gut Microbiome Responses to Paraquat Administration in Male Wistar Rats: Implications for Oxidative Stress.

In this study, we examined the metabolic and gut microbiome responses to paraquat (PQ) in male Wistar rats, focusing on oxidative stress effects. Rats received a single intraperitoneal injection of PQ at 15 and 30 mg/kg, and various oxidative stress parameters (i.e., MDA, SOD, ROS, 8-isoprostanes) were assessed after three days. To explore the omic profile, GC-qTOF and UHPLC-qTOF were performed to assess the plasma metabolome; 1H-NMR was used to assess the urine metabolome; and shotgun metagenomics sequencing was performed to study the gut microbiome. Our results revealed reductions in body weight and tissue changes, particularly in the liver, were observed, suggesting a systemic effect of PQ. Elevated lipid peroxidation and reactive oxygen species levels in the liver and plasma indicated the induction of oxidative stress. Metabolic profiling revealed changes in the tricarboxylic acid cycle, accumulation of ketone body, and altered levels of key metabolites, such as 3-hydroxybutyric acid and serine, suggesting intricate links between energy metabolism and redox reactions. Plasma metabolomic analysis revealed alterations in mitochondrial metabolism, nicotinamide metabolism, and tryptophan degradation. The gut microbiome showed shifts, with higher PQ doses influencing microbial populations (e.g., Escherichia coli and Akkermansia muciniphila) and metagenomic functions (pyruvate metabolism, fermentation, nucleotide and amino acid biosynthesis). Overall, this study provides comprehensive insights into the complex interplay between PQ exposure, metabolic responses, and gut microbiome dynamics. These findings enhance our understanding of the mechanisms behind oxidative stress-induced metabolic alterations and underscore the connections between xenobiotic exposure, gut microbiota, and host metabolism.

Developing a model to predict the early risk of hypertriglyceridemia based on inhibiting lipoprotein lipase (LPL): a translational study.

Hypertriglyceridemia (HTG) is an independent risk factor for atherosclerotic cardiovascular disease (ASCVD). One of the multiple origins of HTG alteration is impaired lipoprotein lipase (LPL) activity, which is an emerging target for HTG treatment. We hypothesised that early, even mild, alterations in LPL activity might result in an identifiable metabolomic signature. The aim of the present study was to assess whether a metabolic signature of altered LPL activity in a preclinical model can be identified in humans. A preclinical LPL-dependent model of HTG was developed using a single intraperitoneal injection of poloxamer 407 (P407) in male Wistar rats. A rat metabolomics signature was identified, which led to a predictive model developed using machine learning techniques. The predictive model was applied to 140 humans classified according to clinical guidelines as (1) normal, less than 1.7 mmol/L; (2) risk of HTG, above 1.7 mmol/L. Injection of P407 in rats induced HTG by effectively inhibiting plasma LPL activity. Significantly responsive metabolites (i.e. specific triacylglycerols, diacylglycerols, phosphatidylcholines, cholesterol esters and lysophospholipids) were used to generate a predictive model. Healthy human volunteers with the impaired predictive LPL signature had statistically higher levels of TG, TC, LDL and APOB than those without the impaired LPL signature. The application of predictive metabolomic models based on mechanistic preclinical research may be considered as a strategy to stratify subjects with HTG of different origins. This approach may be of interest for precision medicine and nutritional approaches.

Mitochondrial dysfunction, lipids metabolism, and amino acid biosynthesis are key pathways for COVID-19 recovery.

The metabolic alterations caused by SARS-CoV-2 infection reflect disease progression. To analyze molecules involved in these metabolic changes, a multiomics study was performed using plasma from 103 patients with different degrees of COVID-19 severity during the evolution of the infection. With the increased severity of COVID-19, changes in circulating proteomic, metabolomic, and lipidomic profiles increased. Notably, the group of severe and critical patients with high HRG and ChoE (20:3) and low alpha-ketoglutaric acid levels had a high chance of unfavorable disease evolution (AUC = 0.925). Consequently, patients with the worst prognosis presented alterations in the TCA cycle (mitochondrial dysfunction), lipid metabolism, amino acid biosynthesis, and coagulation. Our findings increase knowledge regarding how SARS-CoV-2 infection affects different metabolic pathways and help in understanding the future consequences of COVID-19 to identify potential therapeutic targets.

De novo design of energy transfer proteins housing excitonically coupled chlorophyll special pairs.

Natural photosystems couple light harvesting to charge separation using a "special pair" of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independent of complexities of native photosynthetic proteins, and as a first step towards synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that precisely position chlorophyll dimers. X-ray crystallography shows that one designed protein binds two chlorophylls in a binding orientation matching native special pairs, while a second positions them in a previously unseen geometry. Spectroscopy reveals excitonic coupling, and fluorescence lifetime imaging demonstrates energy transfer. We designed special pair proteins to assemble into 24-chlorophyll octahedral nanocages; the design model and cryo-EM structure are nearly identical. The design accuracy and energy transfer function of these special pair proteins suggest that de novo design of artificial photosynthetic systems is within reach of current computational methods.

Brain N-Glycosylation and Lipidomic Profile Changes Induced by a High-Fat Diet in Dyslipidemic Hamsters.

The consumption of diets rich in saturated fats is known to be associated with higher mortality. The adoption of healthy habits, for instance adhering to a Mediterranean diet, has proved to exert a preventive effect towards cardiovascular diseases and dyslipidemia. Little is known about how a suboptimal diet can affect brain function, structure, and the mechanisms involved. The aims of this study were to examine how a high-fat diet can alter the brain N-glycan and lipid profile in male Golden Syrian hamsters and to evaluate the potential of a Mediterranean-like diet to reverse this situation. During twelve weeks, hamsters were fed a normal fat diet (CTRL group), a high-fat diet (HFD group), and a high-fat diet followed by a Mediterranean-like diet (MED group). Out of seventy-two identified N-glycans, fourteen were significant (p < 0.05) between HFD and CTRL groups, nine between MED and CTRL groups, and one between MED and HFD groups. Moreover, forty-nine lipids were altered between HFD and CTRL groups, seven between MED and CTRL groups, and five between MED and HFD groups. Our results suggest that brain N-glycan composition in high-fat diet-fed hamsters can produce events comparable to those found in some neurodegenerative diseases, and may promote brain ageing.

Engineering excitonically coupled dimers in an artificial protein for light harvesting via computational modeling.

In photosynthesis, pigment-protein complexes achieve outstanding photoinduced charge separation efficiencies through a set of strategies in which excited states delocalization over multiple pigments ("excitons") and charge-transfer states play key roles. These concepts, and their implementation in bioinspired artificial systems, are attracting increasing attention due to the vast potential that could be tapped by realizing efficient photochemical reactions. In particular, de novo designed proteins provide a diverse structural toolbox that can be used to manipulate the geometric and electronic properties of bound chromophore molecules. However, achieving excitonic and charge-transfer states requires closely spaced chromophores, a non-trivial aspect since a strong binding with the protein matrix needs to be maintained. Here, we show how a general-purpose artificial protein can be optimized via molecular dynamics simulations to improve its binding capacity of a chlorophyll derivative, achieving complexes in which chromophores form two closely spaced and strongly interacting dimers. Based on spectroscopy results and computational modeling, we demonstrate each dimer is excitonically coupled, and propose they display signatures of charge-transfer state mixing. This work could open new avenues for the rational design of chromophore-protein complexes with advanced functionalities.

Optimization of a GC-MS Injection-Port Derivatization Methodology to Enhance Metabolomics Analysis Throughput in Biological Samples.

Advances in metabolomics analysis and data treatment increase the knowledge of complex biological systems. One of the most used methodologies is gas chromatography-mass spectrometry (GC-MS) due to its robustness, high separation efficiency, and reliable peak identification through curated databases. However, methodologies are not standardized, and the derivatization steps in GC-MS can introduce experimental errors and take considerable time, exposing the samples to degradation. Here, we propose the injection-port derivatization (IPD) methodology to increase the throughput in plasma metabolomics analysis by GC-MS. The IPD method was evaluated and optimized for different families of metabolites (organic acids, amino acids, fatty acids, sugars, sugar phosphates, etc.) in terms of residence time, injection-port temperature, and sample/derivatization reagent ratio. Finally, the method's usefulness was validated in a study consisting of a cohort of obese patients with or without nonalcoholic steatohepatitis. Our results show a fast, reproducible, precise, and reliable method for the analysis of biological samples by GC-MS. Raw data are publicly available at MetaboLights with Study Identifier MTBLS5151.

Surviving on low-energy light comes at a price.

Two species of photosynthetic cyanobacteria can thrive in far-red light but they either become less resilient to photodamage or less energy efficient.

Imbalances in TCA, Short Fatty Acids and One-Carbon Metabolisms as Important Features of Homeostatic Disruption Evidenced by a Multi-Omics Integrative Approach of LPS-Induced Chronic Inflammation in Male Wistar Rats.

Chronic inflammation is an important risk factor in a broad variety of physical and mental disorders leading to highly prevalent non-communicable diseases (NCDs). However, there is a need for a deeper understanding of this condition and its progression to the disease state. For this reason, it is important to define metabolic pathways and complementary biomarkers associated with homeostatic disruption in chronic inflammation. To achieve that, male Wistar rats were subjected to intraperitoneal and intermittent injections with saline solution or increasing lipopolysaccharide (LPS) concentrations (0.5, 5 and 7.5 mg/kg) thrice a week for 31 days. Biochemical and inflammatory parameters were measured at the end of the study. To assess the omics profile, GC-qTOF and UHPLC-qTOF were performed to evaluate plasma metabolome; 1H-NMR was used to evaluate urine metabolome; additionally, shotgun metagenomics sequencing was carried out to characterize the cecum microbiome. The chronicity of inflammation in the study was evaluated by the monitoring of monocyte chemoattractant protein-1 (MCP-1) during the different weeks of the experimental process. At the end of the study, together with the increased levels of MCP-1, levels of interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-α) and prostaglandin E2 (PGE2) along with 8-isoprostanes (an indicative of oxidative stress) were significantly increased (p-value < 0.05). The leading features implicated in the current model were tricarboxylic acid (TCA) cycle intermediates (i.e., alpha-ketoglutarate, aconitic acid, malic acid, fumaric acid and succinic acid); lipids such as specific cholesterol esters (ChoEs), lysophospholipids (LPCs) and phosphatidylcholines (PCs); and glycine, as well as N, N-dimethylglycine, which are related to one-carbon (1C) metabolism. These metabolites point towards mitochondrial metabolism through TCA cycle, ß-oxidation of fatty acids and 1C metabolism as interconnected pathways that could reveal the metabolic effects of chronic inflammation induced by LPS administration. These results provide deeper knowledge concerning the impact of chronic inflammation on the disruption of metabolic homeostasis.

Dynamics of diverse coherences in primary charge separation of bacterial reaction center at 77 K revealed by wavelet analysis.

To uncover the mechanism behind the high photo-electronic conversion efficiency in natural photosynthetic complexes it is essential to trace the dynamics of electronic and vibrational quantum coherences. Here we apply wavelet analysis to two-dimensional electronic spectroscopy data for three purple bacterial reaction centers with mutations that produce drastically different rates of primary charge separation. From the frequency distribution and dynamic evolution features of the quantum beating, electronic coherence with a dephasing lifetime of ~50 fs, vibronic coherence with a lifetime of ~150 fs and vibrational/vibronic coherences with a lifetime of 450 fs are distinguished. We find that they are responsible for, or couple to, different specific steps during the primary charge separation process, i.e., intradimer charge transfer inside the special bacteriochlorophyll pair followed by its relaxation and stabilization of the charge-transfer state. The results enlighten our understanding of how quantum coherences participate in, and contribute to, a biological electron transfer reaction.

Alterations in Metabolome and Microbiome Associated with an Early Stress Stage in Male Wistar Rats: A Multi-Omics Approach.

Stress disorders have dramatically increased in recent decades becoming the most prevalent psychiatric disorder in the United States and Europe. However, the diagnosis of stress disorders is currently based on symptom checklist and psychological questionnaires, thus making the identification of candidate biomarkers necessary to gain better insights into this pathology and its related metabolic alterations. Regarding the identification of potential biomarkers, omic profiling and metabolic footprint arise as promising approaches to recognize early biochemical changes in such disease and provide opportunities for the development of integrative candidate biomarkers. Here, we studied plasma and urine metabolites together with metagenomics in a 3 days Chronic Unpredictable Mild Stress (3d CUMS) animal approach that aims to focus on the early stress period of a well-established depression model. The multi-omics integration showed a profile composed by a signature of eight plasma metabolites, six urine metabolites and five microbes. Specifically, threonic acid, malic acid, alpha-ketoglutarate, succinic acid and cholesterol were proposed as key metabolites that could serve as key potential biomarkers in plasma metabolome of early stages of stress. Such findings targeted the threonic acid metabolism and the tricarboxylic acid (TCA) cycle as important pathways in early stress. Additionally, an increase in opportunistic microbes as virus of the Herpesvirales was observed in the microbiota as an effect of the primary stress stages. Our results provide an experimental biochemical characterization of the early stage of CUMS accompanied by a subsequent omic profiling and a metabolic footprinting that provide potential candidate biomarkers.

Dynamic Stark Effect in Two-Dimensional Spectroscopy Revealing Modulation of Ultrafast Charge Separation in Bacterial Reaction Centers by an Inherent Electric Field.

Despite extensive study, mysteries remain regarding the highly efficient ultrafast charge separation processes in photosynthetic reaction centers (RCs). In this work, transient Stark signals were found to be present in ultrafast two-dimensional electronic spectra recorded for purple bacterial RCs at 77 K. These arose from the electric field that is inherent to the intradimer charge-transfer intermediate of the bacteriochlorophyll pair (P), PA+PB-. By comparing three mutated RCs, a correlation was found between the efficient formation of PA+PB- and a fast charge separation rate. Importantly, the energy level of P* was changed due to the Stark shift, influencing the driving force for P* → P+BA- electron transfer and hence its rate. Furthermore, the orientation and amplitude of the inherent electric field varied in different ways upon different mutation, leading to contrasting changes in the rates. This mechanism of modulation provides a solution to a long-lasting inconsistency between experimental observations and activation energy theory.

Analysis of oxylipins to differentiate between organic and conventional UHT milks.

Nowadays, there is a strong interest in analytical approaches for assessing organic farming practices. Here, we propose that oxylipins, a group of oxidised metabolites derived from various polyunsaturated fatty acids, could be promising biomarkers for organic milk assessment because their biosynthesis is modulated by both precursor fatty acid availability and physiological or pathological status. Thus, we determined 31 fatty acids, 53 triacylglycerols and 37 oxylipins in one hundred commercial UHT milks by chromatographic methods coupled to mass spectrometry. Of these, 52 milks were conventional (34 whole milk, 11 semi-skimmed milk and 7 skimmed milk) and 48 were organic (31 whole milk, 11 semi-skimmed milk and 6 skimmed milk). Several oxylipins (8-HEPE, 5-HEPE, 11-HEPE, 9-HEPE, 18-HEPE, 9-HOTrE, 13-HOTrE, 12,13-DiHODE and 15,16-DiHODE) could distinguish between organic and conventional milks. Within these oxylipins, arachidonic and linoleic acid derived do not correlate with their fatty acid precursors; therefore these oxylipins could be promising as not only diet-dependent biomarkers for organic milk assessment.

Complete mapping of energy transfer pathways in the plant light-harvesting complex Lhca4.

The Lhca4 antenna complex of plant Photosystem I (PSI) is characterized by extremely red-shifted and broadened absorption and emission bands from its low-energy chlorophylls (Chls). The mixing of a charge-transfer (CT) state with the excited state manifold causing these so-called red forms results in highly complicated multi-component excited energy transfer (EET) kinetics within the complex. The two-dimensional electronic spectroscopy experiments presented here reveal that EET towards the CT state occurs on three timescales: fast from the red Chls (within 1 ps), slower (5-7 ps) from the stromal side Chls, and very slow (100-200 ps) from a newly discovered 690 nm luminal trap. The excellent agreement between the experimental data with the previously presented Redfield-Förster exciton model of Lhca4 strongly supports the equilibration scheme of the bulk excitations with the dynamically localized CT on the stromal side. Thus, a complete picture of the energy transfer pathways leading to the population of the CT final trap within the whole Lhca4 complex is presented. In view of the environmental sensitivity of the CT contribution to the Lhca4 energy landscape, we speculate that one role of the CT states is to regulate the EET from the peripheral antenna to the PSI core.

Proteomic Analysis of Heart and Kidney Tissues in Healthy and Metabolic Syndrome Rats after Hesperidin Supplementation.

SCOPE: Proteomics has provided new strategies to elucidate the mechanistic action of hesperidin, a flavonoid present in citrus fruits. Thus, the aim of the present study is to determine the effects of hesperidin supplementation (HS) on the proteomic profiles of heart and kidney tissue samples from healthy and metabolic syndrome (MS) rats. METHODS AND RESULTS: 24 Sprague Dawley rats are randomized into four groups: healthy rats fed with a standard diet without HS, healthy rats administered with HS (100 mg kg-1 day-1 ), MS rats without HS, and MS rats administered with HS (100 mg kg-1 day-1 ) for eight weeks. Heart and kidney samples are obtained, and proteomic analysis is performed by mass spectrometry. Multivariate, univariate, and ingenuity pathways analyses are performed. Comparative and semiquantitative proteomic analyses of heart and kidney tissues reveal differential protein expression between MS rats with and without HS. The top diseases and functions implicated are related to the cardiovascular system, free radical scavenging, lipid metabolism, glucose metabolism, and renal and urological diseases. CONCLUSION: This study is the first to demonstrate the protective capacity of hesperidin to change to the proteomic profiles in relation to different cardiovascular risk biomarkers in the heart and kidney tissues of MS rats.

Cavity-Modified Exciton Dynamics in Photosynthetic Units.

Recently, exciton-photon strong coupling has been proposed as a means to control and enhance energy transfer in ensembles of organic molecules. Here, we demonstrate that the exciton dynamics in an archetypal purple bacterial photosynthetic unit, composed of six LH2 antennas surrounding a single LH1 complex, is greatly modified by its interaction with an optical cavity. We develop a Bloch-Redfield master equation approach that accounts for the interplay between the B800 and B850 bacteriochlorophyll molecules within each LH2 antenna, as well as their interactions with the central LH1 complex. Using a realistic parametrization of both the photosynthetic unit and optical cavity, we investigate the formation of polaritons in the system, revealing that these can be tuned to accelerate its exciton dynamics by 3 orders of magnitude. This yields a significant occupation of the LH1 complex, the stage immediately prior to the reaction center, with only a few-femtosecond delay after the initial excitation of the LH2 B800 pigments. Our theoretical findings unveil polaritonic phenomena as a promising route for the characterization, tailoring, and optimization of light-harvesting mechanisms in natural and artificial photosynthetic processes.

Both electronic and vibrational coherences are involved in primary electron transfer in bacterial reaction center.

Understanding the mechanism behind the near-unity efficiency of primary electron transfer in reaction centers is essential for designing performance-enhanced artificial solar conversion systems to fulfill mankind's growing demands for energy. One of the most important challenges is distinguishing electronic and vibrational coherence and establishing their respective roles during charge separation. In this work we apply two-dimensional electronic spectroscopy to three structurally-modified reaction centers from the purple bacterium Rhodobacter sphaeroides with different primary electron transfer rates. By comparing dynamics and quantum beats, we reveal that an electronic coherence with dephasing lifetime of ~190 fs connects the initial excited state, P*, and the charge-transfer intermediate [Formula: see text]; this [Formula: see text] step is associated with a long-lived quasi-resonant vibrational coherence; and another vibrational coherence is associated with stabilizing the primary photoproduct, [Formula: see text]. The results show that both electronic and vibrational coherences are involved in primary electron transfer process and they correlate with the super-high efficiency.

Vibronic Coherence in the Charge Separation Process of the Rhodobacter sphaeroides Reaction Center.

Two-dimensional electronic spectroscopy was applied to a variant of the reaction center (RC) of purple bacterium Rhodobacter sphaeroides lacking the primary acceptor ubiquinone in order to understand the ultrafast separation and transfer of charge between the bacteriochlorin cofactors. For the first time, characteristic 2D spectra were obtained for the participating excited and charge-transfer states, and the electron-transfer cascade (including two different channels, the P* and B* channels) was fully mapped. By analyzing quantum beats using 2D frequency maps, excited-state vibrational modes at 153 and 33 cm-1 were identified. We speculate that these modes couple to the charge separation (CS) process and collectively optimize the CS and are responsible for the superhigh efficiency.