Metabolic Routes Research Articles

A Dive Into Yeast's Sugar Diet-Comparing the Metabolic Response of Glucose, Fructose, Sucrose, and Maltose Under Dynamic Feast/Famine Conditions.

Microbes experience dynamic conditions in natural habitats as well as in engineered environments, such as large-scale bioreactors, which exhibit increased mixing times and inhomogeneities. While single perturbations have been studied for several organisms and substrates, the impact of recurring short-term perturbations remains largely unknown. In this study, we investigated the response of Saccharomyces cerevisiae to repetitive gradients of four different sugars: glucose, fructose, sucrose, and maltose. Due to different transport mechanisms and metabolic routes, nonglucose sugars lead to varied intracellular responses. To characterize the impact of the carbon sources and the dynamic substrate gradients, we applied both steady-state and dynamic cultivation conditions, comparing the physiology, intracellular metabolome, and proteome. For maltose, the repeated concentration gradients led to a significant decrease in biomass yield. Under glucose, fructose, and sucrose conditions, S. cerevisiae maintained the biomass yield observed under steady-state conditions. Although the physiology was very similar across the different sugars, the intracellular metabolome and proteome were clearly differentiated. Notably, the concentration of upper glycolytic enzymes decreased for glucose and maltose (up to -60% and -40%, respectively), while an increase was observed for sucrose and fructose when exposed to gradients. Nevertheless, for all sugar gradient conditions, a stable energy charge was maintained, ranging between 0.78 and 0.89. This response to maltose is particularly distinct compared to previous single-substrate pulse experiments or limitation to excess shifts, which led to maltose-accelerated death in earlier studies. At the same time, enzymes of lower glycolysis were elevated. Interestingly, common stress-related proteins (GO term: cellular response to oxidative stress) decreased during dynamic conditions.

Metabolomic characteristics of cord blood from neonates with hyperkalemia after antenatal exposure to ritodrine and magnesium sulfate

In the management of pregnancy, ritodrine has been used to prevent preterm birth, and magnesium sulfate (MgSO4) has been used to prevent preterm labor and preeclampsia. Neonates born to mothers receiving these medications occasionally show an increase in serum potassium concentration. Recently, an elevated risk of neonatal hyperkalemia has been reported, particularly when ritodrine and MgSO4 are co-administered; however, the underlying mechanisms remain unclear. We conducted a retrospective cohort study of 142 preterm infants born between 24 and 36 weeks of gestation, categorized into groups exposed to antenatal ritodrine, MgSO4, both agents, or neither. In addition, we investigated the association between potassium levels and metabolites in the serum of umbilical cord blood from 33 infants exposed to antenatal ritodrine and MgSO4 using a metabolomic analysis. Our findings revealed a significant elevation in serum potassium concentration associated with metabolomic findings of activation of glycolysis and the derived metabolic routes in preterm neonates exposed to both ritodrine and MgSO4. Our data indicate that the concurrent administration of ritodrine and MgSO4 caused distinctive metabolic alterations, potentially leading to an additional increase in the intracellular potassium concentration in the fetus. Consequently, this mechanism may imply an elevation in serum potassium concentration postnatally through the redistribution of potassium.

Proteomic Analysis of the Murine Liver Response to Oral Exposure to Aflatoxin B1 and Ochratoxin A: The Protective Role to Bioactive Compounds.

Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) are considered the most important mycotoxins in terms of food safety. The aim of this study was to evaluate the hepatotoxicity of AFB1 and OTA exposure in Wistar rats and to assess the beneficial effect of fermented whey (FW) and pumpkin (P) as functional ingredients through a proteomic approach. For the experimental procedures, rats were fed AFB1 and OTA individually or in combination, with the addition of FW or a FW-P mixture during 28 days. For proteomics analysis, peptides were separated using a LC-MS/MS-QTOF system and differentially expressed proteins (DEPs) were statistically filtered (p < 0.05) distinguishing males from females. Gene ontology visualization allowed the identification of proteins involved in important biological processes such as the response to xenobiotic stimuli and liver development. Likewise, KEGG pathway analysis reported the metabolic routes as the most affected, followed by carbon metabolism and biosynthesis of amino acids. Overall, the results highlighted a strong downregulation of DEPs in the presence of AFB1 and OTA individually but not with the mixture of both, suggesting a synergistic effect. However, FW and P have helped in the mitigation of processes triggered by mycotoxins.

Decarboxylase mediated oxalic acid metabolism is important to antioxidation and detoxification rather than pathogenicity in Magnaporthe oryzae

ABSTRACT Oxalic acid (OA), an essential pathogenic factor, has been identified in several plant pathogens, and researchers are currently pursuing studies on interference with OA metabolism as a treatment for related diseases. However, the metabolic route in Magnaporthe oryzae remains unknown. In this study, we describe D-erythroascorbic acid-mediated OA synthesis and its metabolic and clearance pathways in rice blast fungus. By knocking out the D-arabino-1,4-lactone oxidase gene (Moalo1), one-third of oxalic acid remained in M. oryzae, indicating a main pathway for oxalic acid production. M. oryzae OxdC (MoOxdC) is an oxalate decarboxylase that appears to play a role in relieving oxalic acid toxicity. Loss of Mooxdc does not affect mycelial growth, conidiophore development, or appressorium formation in M. oryzae; however, the antioxidant and pathogenic abilities of the mutant were enhanced. This is owing to Mooxdc deletion upregulated a series of OA metabolic genes, including the oxalate oxidase gene (Mooxo) and Moalo1, as well as both OA transporter genes. Simultaneously, as feedback to the tricarboxylic acid (TCA) cycle, the decrease of formic acid in ΔMooxdc leads to the reduction of acetyl-CoA content, and two genes involved in the β-oxidation of fatty acids were also upregulated, which enhanced the fatty acid metabolism of the ΔMooxdc. Overall, this work reveals the role of OA in M. oryzae. We found that OA metabolism was mainly involved in the growth and development of M. oryzae, OA as a byproduct of D-erythroascorbic acid after removing H2O2, the OA-associated pathway ensures the TCA process and ATP supply.

Advancing Succinic Acid Biomanufacturing Using the Nonconventional Yeast Yarrowia lipolytica.

Succinic acid is an essential bulk chemical with wide-ranging applications in materials, food, and pharmaceuticals. With the advancement of biotechnology, there has been a surge in focus on low-carbon sustainable microbial synthesis methods for producing biobased succinic acid. Due to its high intrinsic acid tolerance, Yarrowia lipolytica has gained recognition as a competitive chassis for the industrial manufacture of succinic acid. This review summarizes the research progress on succinic acid biomanufacturing using Y. lipolytica. First, it introduces the major metabolic routes for succinic acid biosynthesis and the pertinent engineering approaches for building efficient cell factories. Subsequently, we offer a review of methods employed for succinic acid synthesis by Y. lipolytica utilizing alternative substrates as well as the relevant optimization strategies for the fermentation process. Finally, future research directions for improving succinic acid biomanufacturing in Y. lipolytica are delineated in light of the recent progress, obstacles, and trends in this area.

Exploring the functional diversity and metabolic activities of the human gut microbiome in Thai adults in response to a prebiotic diet.

Exploring dietary methods to alter microbial communities and metabolic functions is becoming an increasingly fascinating strategy for improving health. Copra meal hydrolysate (CMH) is alternatively used as a gut health supplement. However, the functional diversity and metabolic activities in gut microbiome in relation to CMH treatment remain largely unknown. Therefore, this study aimed to identify key predominant groups of bacterial species toward diversified metabolic functions, activities, and routes using metaproteomics. As a result, the integrative analysis of metaproteomic data revealed that seven key families across 11 dominant gut bacterial species were concerted. Consistently, across 76,206 proteins assigned to the metabolism of the 255,964 annotated proteins, short-chain fatty acid (SCFA) biosynthesis, lipopolysaccharide (LPS) biosynthesis, and bile acid (BA) metabolism were positively associated with CMH. Further identification of cooperative metabolic routes promisingly highlighted the importance of glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, inositol phosphate metabolism, steroid hormone biosynthesis, O-antigen repeat unit biosynthesis, and chloroalkane and chloroalkene degradation. This work presents an initial study of metaproteomics associated with prebiotic diet in a Thai population-based cohort in a developing Southeast Asian country.IMPORTANCEStudies primarily focused on the impact of CMH on gastrointestinal symptoms and gut microbial compositions. However, as the field moves toward understanding the relationship between microbiome and diet in relation to gut health, it is critical to evaluate how changes in metabolic activities relate to cooperative metabolic routes in the gut microbiome for promoting human health. Through the use of metaproteomics, our findings highlighted the key predominant groups of bacterial species, potential proteins, and their metabolic routes involved in gut metabolism. This study provides comprehensive insights into the fundamental relationship between microbiome and dietary supplements and suggests that metaproteomics is a powerful method for monitoring metabolic functions, activities, and routes in the gut microbiome.

Structural basis of the allosteric regulation of cyanobacterial glucose-6-phosphate dehydrogenase by the redox sensor OpcA

The oxidative pentose phosphate (OPP) pathway is a fundamental carbon catabolic route for generating reducing power and metabolic intermediates for biosynthetic processes. In addition, its first two reactions form the OPP shunt, which replenishes the Calvin-Benson cycle under certain conditions. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting reaction of this metabolic route. In photosynthetic organisms, G6PDH is redox-regulated to allow fine-tuning and to prevent futile cycles while carbon is being fixed. In cyanobacteria, regulation of G6PDH requires the redox protein OpcA, but the underlying molecular mechanisms behind this allosteric activation remain elusive. Here, we used enzymatic assays and in vivo interaction analyses to show that OpcA binds G6PDH under different environmental conditions. However, complex formation enhances G6PDH activity when OpcA is oxidized and inhibits it when OpcA is reduced. To understand the molecular basis of this regulation, we used cryogenic electron microscopy to determine the structure of Synechocystis G6PDH and the G6PDH-OpcA complex. OpcA binds the G6PDH tetramer and induces conformational changes in the active site of G6PDH. The redox sensitivity of OpcA is achieved by intramolecular disulfide bridge formation, which influences the allosteric regulation of G6PDH. In vitro assays reveal that the level of G6PDH activation depends on the number of bound OpcA molecules, which implies that this mechanism allows delicate fine-tuning. Our findings unveil a unique molecular mechanism governing the regulation of the OPP in Synechocystis.

Multiscale interstitial fluid computation modeling of cortical bone to characterize the hydromechanical stimulation of lacunar-canalicular network.

Bone tissue is a biological composite material with a complex hierarchical structure that could continuously adjust its internal structure to adapt to the alterations in the external load environment. The fluid flow within bone is the main route of osteocyte metabolism, and the pore pressure as well as the fluid shear stress generated by it are important mechanical stimuli perceived by osteocytes. Owing to the irregular multiscale structure of bone tissue, the fluid stimulation that lacunar-canalicular network (LCN) in different regions of the tissue underwent remained unclear. In this study, we constructed a multiscale conduction model of fluid flow stimulus signals in bone tissue based on the poroelasticity theory. We analyzed the fluid flow behaviors at the macro-scale (whole bone tissue), macro-meso scale (periosteum, interstitial bone, osteon and endosteum), and micro-scale (lacunar-osteocyte-canalicular) levels. We explored how fluid stimulation at the tissue level correlated with that at the cellular level in cortical bone and characterized the distributions of the pore pressure, fluid velocity and fluid shear stress that the osteocytes experienced across the entire tissue structure. The results showed that the initial conditions of intramedullary pressure had a significant impact on the pore pressure of Haversian systems, but had a relatively small influence on the fluid velocity. The osteocyte which were located at different positions in the bone tissue received very distinct fluid stimuli. Osteocytes in the vicinity of the Haversian Canals experienced higher fluid shear stress stimulation. When the permeability of the LCN was within the range from 10-21m2 to 10-18m2, the distribution of pressure, fluid velocity and fluid shear stress within the osteon near the periosteum and endosteum was significantly different from that in other parts of the bone. However, when the permeability was less than 10-22m2, such a difference did not exist. Particularly, the flow velocity at the lacunae was markedly higher than that in the canaliculi. Meanwhile, the pore pressure and fluid shear stress were conspicuously lower than those in the canaliculi. In this study, we considered the interconnections of different biofunctional units at different scales of bone tissue, construct a more complete multiscale model of bone tissue, and propose that osteocytes at different locations receive different fluid stimuli, which provides a reference for a deeper understanding of bone mechanotransduction.

The preclinical pharmacokinetics of Tolinapant-A dual cIAP1/XIAP antagonist with invivo efficacy.

AT-IAP (1-{6-[(4-fluorophenyl)methyl]-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl}-2-[(2R,5R)-5-methyl-2-{[(3R)-3-methylmorpholin-4-yl]methyl}piperazin-1-yl]ethan-1-one) was identified as a novel potent non-alanine small molecule dual inhibitor of cIAP1/XIAP protein. AT-IAP was assessed in preclinical species, demonstrating favorable bioavailability in rodent species and oral efficacy at 30 mg/kg in MDA-MB-231 mouse xenograft models. The major metabolic route of AT-IAP was identified to be CYP3A driven, resulting in low oral exposure in non-human primate (NHP) studies, given the comparatively high expression of equivalent CYP3A. AT-IAP metabolite identification studies determined ring opening of the morpholino or piperazine moiety. An extensive campaign of optimisation resulted in increased polarity by the addition of a hydroxymethyl, which led to the identification of tolinapant (1-(6-[(4-Fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1 H,2 H,3 H-pyrrolo[3,2- b]pyridin-1-yl)-2-[(2 R,5 R)-5-methyl-2-([(3R)-3-methylmorpholin-4-yl]methyl)piperazin-1-yl]ethan-1-one) with reduced CYP3A metabolism. Tolinapant showed oral bioavailability in rodents and NHP in the range 12-34% at 5 mg/kg. Non-linear pharmacokinetics in NHP were observed at oral doses in the range 5-75 mg/kg. Pharmacodynamic modulation and efficacy were demonstrated in A375 mouse xenograft models at dose ranges between 5 and 20 mg/kg. On-target engagement, as measured by reduction of cIAP 1 protein levels, was confirmed in NHP surrogate tissues and applied to target activity assessments in tolinapant phase1/2 clinical trials.

Proteomic Analysis Unveils the Protective Mechanism of Active Modified Atmosphere Packaging Against Senescence Decay and Respiration in Postharvest Loose-Leaf Lettuce

In this study, physicochemical and proteomic analyses were performed to investigate the effect of modified atmosphere packaging (MAP) on the quality of postharvest loose-leaf lettuce. The results showed that MAP enhanced the sensory characteristics of loose-leaf lettuce and delayed the incidence of postharvest deterioration by suppressing weight loss, electrolyte leakage, and reactive oxygen species levels. MAP-inhibited storage-induced programmed cell death may be attributed to a lower expression of protein disulfide isomerase and a higher expression of oligonucleotide/oligosaccharide binding fold nucleic acid binding site protein and reducing glutamine synthase levels. Also, we explore the potential of MAP to protect against oxidative damage in loose-leaf lettuce by potentially modulating the expression levels of NAC family proteins, which may enhance signaling and the expression of cytochrome c oxidase and membrane-bound pyrophosphate in the oxidative phosphorylation pathway. In addition, MAP potentially delayed postharvest senescence and extended the shelf life of lettuce by regulating key protein metabolic pathways that may reduce respiration rates. These include the NAC family of proteins, enzymes in the oxidative phosphorylation pathway, glutamine synthetize, and other crucial metabolic routes. These findings provide a scientific basis for enhancing the postharvest preservation of leafy vegetables, such as loose-leaf lettuce, through MAP technology.

Metabolic profiling of the synthetic cannabinoid APP-CHMINACA (PX-3) as studied by in vitro and in vivo models.

The metabolic pathways of APP-CHMINACA were characterized to select the markers of intake for implementation into analytical assays used by the clinical and forensic communities. We have combined the evidences obtained by both in vitro experiments and administration studies on mice. APP-CHMINACA was incubated with either human or mouse liver microsomes. Urine and blood samples were collected at different time points from mice after injection of a 3mg/kg dose of the test compound. Samples were analyzed using liquid chromatography-tandem mass spectrometry. The in vitro studies allowed to isolate eight different metabolic reactions, formed by two metabolic routes, with no differences between human and mouse liver microsomes. The main biotransformation route involved the hydrolysis of the distal amide group and the subsequent hydroxylation on the cyclohexyl-methyl ring. The second route involved multiple hydroxylation of the parent compound, followed by reduction to generate minor metabolites. In blood samples, the most abundant substances identified were APP-CHMINACA unchanged and the metabolites formed by the hydrolysis of the distal amide together with its hydroxylated products. In urine samples, four metabolites formed following the hydroxylation of the distal amide hydrolysis metabolite were detected as the most abundant and long-term metabolites. The outcomes of our study showed that the most suitable markers to detect the intake of APP-CHMINACA in blood and urine samples in the framework of toxicological, clinical and forensic investigations were the metabolite formed by the hydrolysis of the distal amide and its hydroxylated products.

Application of 1-octanol in the extraction and GC-FID analysis of volatile organic compounds produced in biogas and biohydrogen processes

Information about the volatile organic compounds generated in biogas and hydrogen production bioreactors is essential to elucidate the metabolic routes and varying yields of CH4 and H2 processes. In this work, the determination of 12 compounds (acetone, methanol, ethanol, 1-propanol, 1-butanol, and acetic, propanoic, butyric, isovaleric, valeric, caproic, and lactic acids) was performed by gas chromatography, after a vortex-assisted liquid–liquid microextraction (VALLME) procedure using 1.2 mL of crude sample and 400 µL of 1-octanol. Optimization of the separation process was performed, considering the solvent viscosity. The analytical curves were validated using ANOVA, demonstrating satisfactory precision and accuracy. Selectivity was confirmed by GC–MS analysis, which allowed the detection of glycerol, 1,3-propanediol, and 1,3-butanediol in some samples. Methanol levels exceeded the upper limit of quantification, with acetic acid and ethanol being the predominant compounds in the analyzed reactors. An additional investigation was conducted to assess potential interferences for lactic acid. The developed method employs a biodegradable extraction solvent, without any need for a dispersing solvent, and involves a single chromatographic run, without any derivatization steps.

Exploring microbial diversity and functionality in Verdejo wine vinegar fermentation through LC-MS/MS analysis

The identification of microorganisms in vinegar production remains a challenge mainly, due to the difficulty of isolating and characterizing the microbiota involved. This implies that despite progress in understanding the composition and role of these microbial communities, there are still virtually unknown microorganisms in these environments. Metaproteomics reveals microbial diversity and behavior by analysis of proteins, offering precise insights into key moments and conditions in biological processes like acetification. This study consists of a thorough metaproteomic analysis during the elaboration of an innovative vinegar, Verdejo wine vinegar. Acetic acid fermentation occurred by submerged culture in an 8L automated Frings reactor operated in semi-continuous mode. LC-MS/MS identified 1626 proteins from 149 taxonomic groups, mainly Acetobacteraceae (1529), followed by yeast (47), lactic acid bacteria (29), and Archaea (21). Komagataeibacter (73.7%), Acetobacter (10.7%), Gluconacetobacter (1.7%), Novacetimonas (1.6%), and Gluconobacter (1.3%) were prevalent genera, with K. europaeus being the dominant species (56.7%). To our knowledge, this is the first metaproteomic approach to comprehensively address the study of so diverse microbial populations. GO Term analysis emphasized "heterocyclic compound binding" and "organic substance metabolic process", while analysis of protein-protein interactions identified key metabolic processes, such as amino acid biosynthesis, especially BCAAs, and crucial energetic pathways such as the TCA cycle. Stress response mechanisms, such as the dnaK-dnaJ-grpE and cl2p system, have been also highlighted. These analyses revealed the molecular and functional complexity of the acetification process, highlighting the critical importance of diverse metabolic routes in the adaptation of the microbiota members to the medium conditions. Overall, this study aims to characterize the microbiota driving Verdejo wine acetification and explore its composition, functions, and key metabolic processes.

Inhibition of the miR-1914-5p increases the oxidative metabolism in cellular model of steatosis by modulating the Sirt1-PGC-1α pathway and systemic cellular activity.

Metabolic associated fatty liver disease (MAFLD) is considered an indicator of metabolic syndrome, which affects millions of people around the world and no effective treatment is currently available. MAFLD involves a wide spectrum of liver damage, that initiates from steatosis (fatty live) and may progress to more complex pathophysiology. Then, details in lipid metabolism controlling should be explored aiming to control the fatty liver. In this context, the miR-1914-5p can be considered a potential biotechnology tool to control lipid metabolism in hepatic cells. This miRNA finds potential mRNA binding sequences in more than 100 molecules correlated with energy production and lipid metabolism pointed in bioinformatic platforms. The present study addressed the miR-1914-5p effects in hepatic HepG2/LX-2 co-cultured cells in a in vitro steatotic environment stablished by the addition of 400 μM of a mixture of oleic and palmitic acids. The analyses demonstrated that the inhibition of the miRNA reduced energetic metabolites such as total lipids, triglycerides, cholesterol and even glucose. In addition, the miR-inhibitor-transfected cells did not present any deleterious effect in cellular environment by controlling reactive oxygen species production (ROS), mitochondrial membrane potential (ΔΨm) and even the pro-inflammatory environment. Moreover, the functional effect of the investigated miR, suggested its close connection to the modulation of Sirt-1-PGC1-α pathway, a master switch metabolic route that controlls cellular energetic metabolism. Our assays also suggested a synergistic effect of this miR-1914-5p in cell metabolism, which should be considered as a strong candidate to control steatotic environment in future clinical trials.

The biochemical mechanisms of plastic biodegradation.

Since the invention of the first synthetic plastic, an estimated 12 billion metric tons of plastics have been manufactured, 70% of which was produced in the last 20 years. Plastic waste is placing new selective pressures on humans and the organisms we depend on, yet it also places new pressures on microorganisms as they compete to exploit this new and growing source of carbon. The limited efficacy of traditional recycling methods on plastic waste, which can leach into the environment at low purity and concentration, indicates the utility of this evolving metabolic activity. This review will categorize and discuss the probable metabolic routes for each industrially relevant plastic, rank the most effective biodegraders for each plastic by harmonizing and reinterpreting prior literature, and explain the experimental techniques most often used in plastic biodegradation research, thus providing a comprehensive resource for researchers investigating and engineering plastic biodegradation.

Pharmacokinetics and ADME Profiling of Tanimilast Following an Intravenous 14C-Microtracer co-administered with an Inhaled Dose in Healthy Male Individuals.

Tanimilast is an inhaled phosphodiesterase-4 inhibitor currently in phase 3 clinical development for treating chronic obstructive pulmonary disease (COPD) and asthma. This trial aimed to characterize the pharmacokinetics, mass balance, and metabolite profiling of tanimilast. Eight healthy male volunteers received a single dose of non-radiolabeled tanimilast via powder inhaler (NEXThaler® (3200μg)), followed by a concomitant intravenous (IV) infusion of a microtracer ([14C]-tanimilast: 18.5μg and 500nCi). Plasma, whole blood, urine, and feces samples were collected up to 240 hours post-dose to quantify non-radiolabeled tanimilast, [14C]-tanimilast, and total-[14C]. The inhaled absolute bioavailability of tanimilast was found to be approximately 50%. Following IV administration of [14C]-tanimilast, plasma clearance was 22 L/h, the steady-state volume of distribution was 201 L, and the half-life was shorter compared to inhaled administration (14 vs. 39 hours, respectively), suggesting that plasma elimination is limited by the absorption rate from the lungs. 79% (71% in feces; 8% in urine) of the IV dose was recovered in excreta as total-[14C]. [14C]-tanimilast was the major radioactive compound in plasma, while no recovery was observed in urine and only 0.3% was recovered in feces, indicating predominant elimination through metabolic route. Importantly, as far as no metabolites accounting for more than 10% of the circulating drug-related exposure in plasma or the administered dose in excreta were detected, no further qualification is required according to regulatory guidelines. This study design successfully characterized the absorption, distribution, and elimination of tanimilast, providing key pharmacokinetic parameters to support its clinical development and regulatory application. Significance Statement This trial investigates PK and ADME profile of tanimilast, an inhaled PDE4 inhibitor for COPD and asthma. Eight male volunteers received a dose of non-radiolabeled tanimilast via NEXThaler® and a microtracer IV dose. Results show pivotal PK results for the characterization of tanimilast, excretion route and quantification of significant metabolites, facilitating streamlined clinical development and regulatory approval.

Towards a complete description of the reaction mechanisms between nitrenium ions and water.

Nitrenium ions are intermediates in the metabolic routes producing the highly carcinogenic nitrosamines and binding to DNA molecules. The reaction mechanism of nitrenium molecules with explicit water molecules is sensibly dependent on the number of waters: when a second molecule is involved, it acts as a catalyst for the reaction, lowering intrinsic activation barriers regardless of the substituent. For all cases, the reaction force constants and reaction electron flux indicate highly synchronous reactions for . Conversely, for highly non-synchronous reactions are obtained, involving two separate proton transfers happening early and late in the reaction path. As a test case, for the simplest reactions, orbital interactions within the NBO paradigm, bond orders, and their derivatives indicate that each individual proton transfer is highly synchronous. Molecular geometries were optimized and characterized at the B3LYP/6-311++G(d, p) level. Intrinsic reaction coordinates were calculated. CCSD(T) single point energies with the same basis were computed on all stationary points. The reaction force, reaction force constant, and reaction electron flux are used to study the evolution of the reacting systems. Natural bond orbitals are used to understand the primitive changes driving the reaction.

Salinity influence on Mytilus galloprovincialis exposed to antineoplastic agents: a transcriptomic, biochemical, and histopathological approach

Nowadays, aquatic species face a variety of environmental risks associated with pharmaceutical consumption. More specifically, the increased number of cancer patients has been accompanied by an increased consumption of antineoplastic drugs, such as ifosfamide (IF) and cyclophosphamide (CP). These drugs have been found in aquatic ecosystems, raising concerns about their impact, especially on estuarine species, as marine waters are the final recipients of continental effluents. Simultaneously, predicted climatic changes, such as salinity shifts, may threaten organisms. Considering this, the present research aims to investigate the combined effects of IF and CP, and salinity shifts. For this, a transcriptomic, biochemical, and histopathological assessment was made using the bivalve species Mytilus galloprovincialis exposed for 28 days to IF and CP (500 ng/L), individually, at different salinity levels (20, 30, and 40). IF and CP up-regulated metabolism-related gene cyp3a1, with CP also affecting abcc gene, showing minimal salinity impact and highlighting the importance of these metabolic routes in mussels. Salinity shifts affected the transcription of genes related to apoptosis and cell cycle growth, such as p53, as well as the aerobic metabolism, the antioxidant and biotransformation mechanisms. These findings indicate mussels' high metabolic adaptability to osmotic stress. Under CP exposure and low salinity, mussels exhibited increased cellular damage and histopathological effects in digestive gland tubules, revealing detrimental effects towards M. galloprovincialis, and suggesting that a metabolic slowdown and activation of antioxidant mechanisms helped prevent oxidative damage at the control and high salinities. Overall, results reinforce the need for antineoplastics ecotoxicological risk assessment, especially under foreseen climate change scenarios.

The β-Ketoacyl-ACP Synthase FabF Catalyzes Carbon-Carbon Bond Formation in a Bimodal Pattern for Fatty Acid Biosynthesis.

Fatty acids produced by the type-II fatty acid biosynthetic pathway (FAS-II) are essential biomaterials for bacterial membrane construction and numerous metabolic routes. The β-ketoacyl-ACP synthase FabF catalyzes the key C-C bond formation step for fatty acid elongation in FAS-II. Here, we revealed the substrate recognition and catalytic mechanisms of FabF by determining FabF-ACP complexes. FabF displays a distinctive bimodal catalytic pattern specifically on C6 and C10 acyl-ACP substrates. It utilizes positively charged residues located on the η3-helix and loop1 regions near the catalytic tunnel entrance to bind ACP, and two hydrophobic cavities as well as "front", "middle", and "back" door residues to specifically stabilize C6 and C10 acyl substrates for preferential catalysis. Further quantum chemistry calculations suggest that the FabF catalytic residues Lys336 and His304 facilitate proton transfer during condensation catalysis and C-C bond formation. Our results provide key mechanistic insights into the biosynthesis of molecular carbon skeletons based on ketosynthases that are highly conserved through the FAS and polyketide synthase (PKS) analogous biosynthetic routes, broaden the understanding of the tricarboxylic acid cycle that utilizes lipoic acid derived from C8-ACP accumulated due to the FabF distinctive catalytic pattern for oxidative decarboxylations, and may facilitate the development of narrow-spectrum antibacterial drugs.

The β‐Ketoacyl‐ACP Synthase FabF Catalyzes Carbon‐Carbon Bond Formation in a Bimodal Pattern for Fatty Acid Biosynthesis

AbstractFatty acids produced by the type‐II fatty acid biosynthetic pathway (FAS‐II) are essential biomaterials for bacterial membrane construction and numerous metabolic routes. The β‐ketoacyl‐ACP synthase FabF catalyzes the key C−C bond formation step for fatty acid elongation in FAS‐II. Here, we revealed the substrate recognition and catalytic mechanisms of FabF by determining FabF‐ACP complexes. FabF displays a distinctive bimodal catalytic pattern specifically on C6 and C10 acyl‐ACP substrates. It utilizes positively charged residues located on the η3‐helix and loop1 regions near the catalytic tunnel entrance to bind ACP, and two hydrophobic cavities as well as “front”, “middle”, and “back” door residues to specifically stabilize C6 and C10 acyl substrates for preferential catalysis. Further quantum chemistry calculations suggest that the FabF catalytic residues Lys336 and His304 facilitate proton transfer during condensation catalysis and C−C bond formation. Our results provide key mechanistic insights into the biosynthesis of molecular carbon skeletons based on ketosynthases that are highly conserved through the FAS and polyketide synthase (PKS) analogous biosynthetic routes, broaden the understanding of the tricarboxylic acid cycle that utilizes lipoic acid derived from C8‐ACP accumulated due to the FabF distinctive catalytic pattern for oxidative decarboxylations, and may facilitate the development of narrow‐spectrum antibacterial drugs.