Antibiotic class with potent in vivo activity targeting lipopolysaccharide synthesis in Gram-negative bacteria.

Here, we describe the identification of an antibiotic class acting via LpxH, a clinically unexploited target in lipopolysaccharide synthesis. The lipopolysaccharide synthesis pathway is essential in most Gram-negative bacteria and there is no analogous pathway in humans. Based on a series of phenotypic screens, we identified a hit targeting this pathway that had activity on efflux-defective strains of Escherichia coli. We recognized common structural elements between this hit and a previously published inhibitor, also with activity against efflux-deficient bacteria. With the help of X-ray structures, this information was used to design inhibitors with activity on efflux-proficient, wild-type strains. Optimization of properties such as solubility, metabolic stability and serum protein binding resulted in compounds having potent in vivo efficacy against bloodstream infections caused by the critical Gram-negative pathogens E. coli and Klebsiella pneumoniae. Other favorable properties of the series include a lack of pre-existing resistance in clinical isolates, and no loss of activity against strains expressing extended-spectrum-ß-lactamase, metallo-ß-lactamase, or carbapenemase-resistance genes. Further development of this class of antibiotics could make an important contribution to the ongoing struggle against antibiotic resistance.

3-(Adenosylthio)benzoic Acid Derivatives as SARS-CoV-2 Nsp14 Methyltransferase Inhibitors.

SARS-CoV-2 nsp14 guanine-N7-methyltransferase plays an important role in the viral RNA translation process by catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to viral mRNA cap. We report a structure-guided design and synthesis of 3-(adenosylthio)benzoic acid derivatives as nsp14 methyltransferase inhibitors resulting in compound 5p with subnanomolar inhibitory activity and improved cell membrane permeability in comparison with the parent inhibitor. Compound 5p acts as a bisubstrate inhibitor targeting both SAM and mRNA-binding pockets of nsp14. While the selectivity of 3-(adenosylthio)benzoic acid derivatives against human glycine N-methyltransferase was not improved, the discovery of phenyl-substituted analogs 5p,t may contribute to further development of SARS-CoV-2 nsp14 bisubstrate inhibitors.

Reduced GFAP Expression in Bergmann Glial Cells in the Cerebellum of Sigma-1 Receptor Knockout Mice Determines the Neurobehavioral Outcomes after Traumatic Brain Injury.

Neuroprotective effects of Sigma-1 receptor (S1R) ligands have been observed in multiple animal models of neurodegenerative diseases. Traumatic brain injury (TBI)-related neurodegeneration can induce long-lasting physical, cognitive, and behavioral disabilities. The aim of our study was to evaluate the role of S1R in the development of neurological deficits after TBI. Adult male wild-type CD-1 (WT) and S1R knockout (S1R-/-) mice were subjected to lateral fluid percussion injury, and behavioral and histological outcomes were assessed for up to 12 months postinjury. Neurological deficits and motor coordination impairment were less pronounced in S1R-/- mice with TBI than in WT mice with TBI 24 h after injury. TBI-induced short-term memory impairments were present in WT but not S1R-/- mice 7 months after injury. Compared to WT animals, S1R-/- mice exhibited better motor coordination and less pronounced despair behavior for up to 12 months postinjury. TBI induced astrocyte activation in the cortex of WT but not S1R-/- mice. S1R-/- mice presented a significantly reduced GFAP expression in Bergmann glial cells in the molecular layer of the cerebellum compared to WT mice. Our findings suggest that S1R deficiency reduces TBI-induced motor coordination impairments by reducing GFAP expression in Bergmann glial cells in the cerebellum.

Neuroprotective and anti-inflammatory activity of DAT inhibitor R-phenylpiracetam in experimental models of inflammation in male mice.

R-phenylpiracetam (R-PhP, (4R)-2-(4-phenyl-2-oxopyrrolidin-1-yl)acetamide) is an optical isomer of phenotropil, a clinically-used nootropic drug that improves physical condition and cognition. Recently, R-PhP was shown to bind to the dopamine transporter (DAT). Since growing evidence suggests that dysfunction of the dopaminergic system is associated with persistent neuroinflammation, the aim of this study was to determine whether R-PhP, an inhibitor of DAT, has neuroprotective and anti-inflammatory effects in male mice. The pharmacokinetic profiles of R-PhP in mouse plasma and its bioavailability in brain tissue were assessed. To study possible molecular mechanisms involved in the anti-inflammatory activity of R-PhP, target profiling was performed using radioligand binding and enzymatic activity assays. To clarify the neuroprotective and anti-inflammatory effects of R-PhP, we used a lipopolysaccharide (LPS)-induced endotoxaemia model characterized by reduced body temperature and overexpression of inflammatory genes in the brain. In addition, the antinociceptive and anti-inflammatory effects of R-PhP were tested using carrageenan-induced paw oedema and formalin-induced paw-licking tests. R-PhP (50 mg/kg) reached the brain tissue 15 min after intraperitoneal (ip) and peroral (po) injections. The maximal concentration of R-PhP in the brain tissues was 28 µg/g and 18 µg/g tissue after ip and po administration, respectively. In radioligand binding assays, DAT was the only significant molecular target found for R-PhP. A single ip injection of R-PhP significantly attenuated the LPS-induced body temperature reduction and the overexpression of inflammatory genes, such as tumour necrosis factor-α (TNF-α), interleukin 1 beta (IL-1ß) and inducible nitric oxide synthase (iNOS). Seven-day po pretreatment with R-PhP dose-dependently reduced paw oedema and the antinociceptive response, as shown by the carrageenan-induced paw oedema test. In addition, R-PhP decreased the nociceptive response during the inflammatory phase in the formalin-induced paw-licking test. Our study showed that R-PhP possesses neuroprotective and anti-inflammatory effects, demonstrating the potential of DAT inhibitors as effective therapeutics.

L-Carnitine Supplementation Increases Trimethylamine-N-Oxide but not Markers of Atherosclerosis in Healthy Aged Women.

BACKGROUND: L-carnitine can be metabolized to trimethylamine N-oxide (TMAO), a molecule that promotes atherogenesis through its interaction with macrophages and lipid metabolism. OBJECTIVE: The aim of the present study was to assess whether L-carnitine supplementation may promote changes in selected serum biomarkers of atherosclerosis. METHODS: Before the start, in the mid-point and after completing the 24-weeks supplementation protocol, fasting blood samples were taken from the antecubital vein. Plasma free L-carnitine and TMAO were determined by the UPLC/MS/MS method. Serum proteins were determined by the enzyme immunoassay method using commercially available kits. Total cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, and triglycerides have been determined using standard automatic analyzer. RESULTS: L-carnitine supplementation elevated fasting plasma carnitine in the mid-point of our study and it remained increased until the end of supplementation period. Moreover, it induced tenfold increase in plasma TMAO concentration but did not affect serum C-reactive protein, interleukin-6, tumour necrosis factor-α, L-selectin, P-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1 or lipid profile markers. CONCLUSION: We demonstrated that -although oral L-carnitine supplementation significantly -increased plasma TMAO concentration, no lipid profile changes or other markers of adverse cardiovascular events were detected in healthy aged women over the period of 24 weeks.

CntA oxygenase substrate profile comparison and oxygen dependency of TMA production in Providencia rettgeri.

CntA oxygenase is a Rieske 2S-2Fe cluster-containing protein that has been previously described as able to produce trimethylamine (TMA) from carnitine, gamma-butyrobetaine, glycine betaine, and in one case, choline. TMA found in humans is exclusively of bacterial origin, and its metabolite, trimethylamine oxide (TMAO), has been associated with atherosclerosis and heart and renal failure. We isolated four different Rieske oxygenases and determined that there are no significant differences in their substrate panels. All three had high activity toward carnitine/gamma-butyrobetaine, medium activity toward glycine betaine, and very low activity toward choline. We tested the influence of low oxygen concentrations on TMA production in CntA-containing Providencia rettgeri cell cultures and discovered that this process, although dependent on the amount of oxygen, is still feasible in environments with 1 and 0.2% oxygen, which is comparable to oxygen levels in some parts of the digestive system.

Structure and Function of CutC Choline Lyase from Human Microbiota Bacterium Klebsiella pneumoniae.

CutC choline trimethylamine-lyase is an anaerobic bacterial glycyl radical enzyme (GRE) that cleaves choline to produce trimethylamine (TMA) and acetaldehyde. In humans, TMA is produced exclusively by the intestinal microbiota, and its metabolite, trimethylamine oxide, has been associated with a higher risk of cardiovascular diseases. Therefore, information about the three-dimensional structures of TMA-producing enzymes is important for microbiota-targeted drug discovery. We have cloned, expressed, and purified the CutC GRE and the activating enzyme CutD from Klebsiella pneumoniae, a representative of the human microbiota. We have determined the first crystal structures of both the choline-bound and choline-free forms of CutC and have discovered that binding of choline at the ligand-binding site triggers conformational changes in the enzyme structure, a feature that has not been observed for any other characterized GRE.

Decreased acylcarnitine content improves insulin sensitivity in experimental mice models of insulin resistance.

The important pathological consequences of insulin resistance arise from the detrimental effects of accumulated long-chain fatty acids and their respective acylcarnitines. The aim of this study was to test whether exercise combined with decreasing the content of long-chain acylcarnitines represents an effective strategy to improve insulin sensitivity in diabetes. We used a novel compound, 4-[ethyl(dimethyl)ammonio]butanoate (methyl-GBB), treatment and exercise to decrease acylcarnitine contents in the plasma and muscles in the insulin resistance models of high fat diet (HFD) fed C57BL/6 mice and db/db mice. The methyl-GBB treatment induced a substantial decrease in all acylcarnitine concentrations in both fed and fasted states as well as when it was combined with exercise. In the HFD fed mice methyl-GBB treatment improved both glucose and insulin tolerance. Methyl-GBB administration, exercise and the combination of both improved insulin sensitivity and reduced blood glucose levels in db/db mice. Methyl-GBB administration and the combination of the drug and exercise activated the PPARα/PGC1α signaling pathway and stimulated the corresponding target gene expression. Insulin insensitivity in db/db mice was not induced by significantly increased fatty acid metabolism, while increased insulin sensitivity by both treatments was not related to decreased fatty acid metabolism in muscles. The pharmacologically reduced long-chain acylcarnitine content represents an effective strategy to improve insulin sensitivity. The methyl-GBB treatment and lifestyle changes via increased physical activity for one hour a day have additive insulin sensitizing effects in db/db mice.

Determination of trimethylamine-N-oxide in combination with L-carnitine and γ-butyrobetaine in human plasma by UPLC/MS/MS.

An ultra-high-performance liquid chromatography-mass spectrometry (UPLC/MS/MS) method was developed and validated for the quantification of trimethylamine-N-oxide (TMAO) simultaneously with TMAO-related molecules L-carnitine and γ-butyrobetaine (GBB) in human blood plasma. The separation of analytes was achieved using a Hydrophilic interaction liquid chromatography (HILIC)-type column with ammonium acetate-acetonitrile as the mobile phase. TMAO determination was validated according to valid US Food and Drug Administration guidelines. The developed method was successfully applied to plasma samples from healthy volunteers.

Expression and purification of active, stabilized trimethyllysine hydroxylase.

Trimethyllysine hydroxylase (TMLH) catalyses the first step in carnitine biosynthesis - the conversion of N6,N6,N6-trimethyl-l-lysine to 3-hydroxy-N6,N6,N6-trimethyl-l-lysine. By changing carnitine availability it is possible to optimise cardiac energy metabolism, that is beneficial under certain ischemic conditions. Previous efforts have been devoted towards the inhibition of gamma-butyrobetaine dioxygenase, which catalyses the last step in carnitine biosynthesis. However, the effects of TMLH activity regulation are currently unexplored. To facilitate the development of specific ligands of TMLH, large quantities of recombinant protein are necessary for downstream binding and structural studies. Here, we describe an efficient system for expressing and purifying active and stable TMLH as a maltose-binding protein fusion in Escherichiacoli.

Long-chain acylcarnitine content determines the pattern of energy metabolism in cardiac mitochondria.

In the heart, a nutritional state (fed or fasted) is characterized by a unique energy metabolism pattern determined by the availability of substrates. Increased availability of acylcarnitines has been associated with decreased glucose utilization; however, the effects of long-chain acylcarnitines on glucose metabolism have not been previously studied. We tested how changes in long-chain acylcarnitine content regulate the metabolism of glucose and long-chain fatty acids in cardiac mitochondria in fed and fasted states. We examined the concentrations of metabolic intermediates in plasma and cardiac tissues under fed and fasted states. The effects of substrate availability and their competition for energy production at the mitochondrial level were studied in isolated rat cardiac mitochondria. The availability of long-chain acylcarnitines in plasma reflected their content in cardiac tissue in the fed and fasted states, and acylcarnitine content in the heart was fivefold higher in fasted state compared to the fed state. In substrate competition experiments, pyruvate and fatty acid metabolites effectively competed for the energy production pathway; however, only the physiological content of acylcarnitine significantly reduced pyruvate and lactate oxidation in mitochondria. The increased availability of long-chain acylcarnitine significantly reduced glucose utilization in isolated rat heart model and in vivo. Our results demonstrate that changes in long-chain acylcarnitine contents could orchestrate the interplay between the metabolism of pyruvate-lactate and long-chain fatty acids, and thus determine the pattern of energy metabolism in cardiac mitochondria.

Selective inhibition of OCTN2 is more effective than inhibition of gamma-butyrobetaine dioxygenase to decrease the availability of l-carnitine and to reduce myocardial infarct size.

l-Carnitine is a cofactor in the energy metabolism pathways where it drives the uptake and oxidation of long chain fatty acids (LCFA) by mitochondria. LCFA lipotoxicity causes mitochondrial damage and results in an insufficient energy supply and a decrease in l-carnitine content limits LCFA flux and protects mitochondria. Here, we tested whether the inhibition of GBB dioxygenase (BBOX) or organic cation transporter 2 (OCTN2) is the most effective strategy to decrease l-carnitine content. The activity of 51 compounds was tested and we identified selective inhibitors of OCTN2. In contrast to selective inhibitors of BBOX, OCTN2 inhibitors induced a 10-fold decrease in l-carnitine content in the heart tissues and a significant 35% reduction of myocardial infarct size. In addition, OCTN2 inhibition correlated with the inhibitor content in the heart tissues, and OCTN2 could potentially be an efficient target to increase drug transport into tissues and to reduce drug elimination by urine. In conclusion, the results of this study confirm that selective inhibition of OCTN2, compared to selective inhibition of BBOX, is a far more effective approach to decrease l-carnitine content and to induce cardioprotective effects. OCTN2 could potentially be an efficient tool to increase drug transport in tissues and to reduce drug elimination via urine.

Innovative Approach to Enhance Bioavailability of Birch Bark Extracts: Novel Method of Oleogel Development Contrasted with Other Dispersed Systems.

Birch outer bark extract (BBE), containing pentacyclic triterpenes such as betulin, lupeol, and betulinic acid, is a widely recognized natural product renowned for its diverse pharmacological effects. However, its limited water solubility restricts its bioavailability. Therefore, the main objective is to enhance the bioavailability of BBE for pharmaceutical use. In this study, we aimed to develop a dispersion system utilizing a unique oleogel-producing method through the recrystallization of BBE from an ethanol solution in the oil phase. We generated an oleogel that demonstrates a notable 42-80-fold improvement in betulin and lupeol peroral bioavailability from BBE in Wistar rats, respectively. A physical paste-like BBE hydrogel developed with antisolvent precipitation showed a 16-56-fold increase in the bioavailability of betulin and lupeol from BBE in rat blood plasma, respectively. We also observed that the repeated administration of the BBE oleogel did not exhibit any toxicity at the tested dose (38.5 mg/kg betulin, 5.2 mg/kg lupeol, 1.5 mg/kg betulinic acid daily for 7 days). Betulin and betulinic acid were not detected in rat heart, liver, kidney, or brain tissues after the peroral administration of the oleogel daily for 7 days. Lupeol was found in rat heart, liver, and kidney tissues.

Effect of NAT2, GSTM1 and CYP2E1 genetic polymorphisms on plasma concentration of isoniazid and its metabolites in patients with tuberculosis, and the assessment of exposure-response relationships.

Objectives: Isoniazid is a key drug in the chemotherapy of tuberculosis (TB), however, interindividual variability in pharmacokinetic parameters and drug plasma levels may affect drug responses including drug induced hepatotoxicity. The current study investigated the relationships between isoniazid exposure and isoniazid metabolism-related genetic factors in the context of occurrence of drug induced hepatotoxicity and TB treatment outcomes. Methods: Demographic characteristics and clinical information were collected in a prospective TB cohort study in Latvia (N = 34). Time to sputum culture conversion (tSCC) was used as a treatment response marker. Blood plasma concentrations of isoniazid (INH) and its metabolites acetylisoniazid (AcINH) and isonicotinic acid (INA) were determined at three time points (pre-dose (0 h), 2 h and 6 h after drug intake) using liquid chromatography-tandem mass spectrometry. Genetic variations of three key INH-metabolizing enzymes (NAT2, CYP2E1, and GSTM1) were investigated by application PCR- and Next-generation sequencing-based methods. Depending on variables, group comparisons were performed by Student's t-test, one-way ANOVA, Mann-Whitney-Wilcoxon, and Kruskal-Wallis tests. Pearson correlation coefficient was calculated for the pairs of normally distributed variables; model with rank transformations were used for non-normally distributed variables. Time-to-event analysis was performed to analyze the tSCC data. The cumulative probability of tSCC was obtained using Kaplan-Meier estimators. Cox proportional hazards models were fitted to estimate hazard rate ratios of successful tSCC. Results: High TB treatment success rate (94.1%) was achieved despite the variability in INH exposure. Clinical and demographic factors were not associated with either tSCC, hepatotoxicity, or INH pharmacokinetics parameters. Correlations between plasma concentrations of INH and its metabolites were NAT2 phenotype-dependent, while GSTM1 genetic variants did not showed any effects. CYP2E1*6 (T > A) allelic variant was associated with INH pharmacokinetic parameters. Decreased level of AcINH was associated with hepatotoxicity, while decreased values of INA/INH and AcINH/INH were associated with month two sputum culture positivity. Conclusion: Our findings suggest that CYP2E1, but not GSTM1, significantly affects the INH pharmacokinetics along with NAT2. AcINH plasma level could serve as a biomarker for INH-related hepatotoxicity, and the inclusion of INH metabolite screening in TB therapeutic drug monitoring could be beneficial in clinical studies for determination of optimal dosing strategies.

Exploring Variability in Rifampicin Plasma Exposure and Development of Anti-Tuberculosis Drug-Induced Liver Injury among Patients with Pulmonary Tuberculosis from the Pharmacogenetic Perspective.

Genetic polymorphisms can exert a considerable impact on drug pharmacokinetics (PK) and the development of adverse drug reactions (ADR). However, the effect of genetic polymorphisms on the anti-tuberculosis (anti-TB) drug, and particularly rifampicin (RIF), exposure or anti-TB drug-induced liver injury (DILI) remains uncertain. Here, we evaluated the relationship between single nucleotide polymorphisms (SNPs) detected in the RIF pharmacogenes (AADAC, SLCO1B1, SLCO1B3, ABCB1, and NR1I2) and RIF PK parameters, as well as anti-TB treatment-associated DILI. In total, the study enrolled 46 patients with drug-susceptible pulmonary TB. The RIF plasma concentration was measured using the LC-MS/MS method in the blood samples collected pre-dose and 2 and 6 h post-dose, whilst the DILI status was established using the results from blood biochemical analysis performed before and 10-12 days after treatment onset. The genotyping was conducted using a targeted NGS approach. After adjustment for confounders, the patients carrying the rs3732357 GA/AA genotype of the NR1I2 gene were found to have significantly lower RIF plasma AUC0-6 h in comparison to those with GG genotype, while the difference in RIF plasma Cmax was insignificant. None of the analyzed SNPs was related to DILI. Hence, we are the first to report NR1I2 intronic SNP rs3732357 as the genetic component of variability in RIF exposure. Regarding anti-TB treatment-associated DILI, the other preexisting factors promoting this ADR should be considered.

Hydroxymethylglutaryl-CoA reductase activity is essential for mitochondrial ß-oxidation of fatty acids to prevent lethal accumulation of long-chain acylcarnitines in the mouse liver.

BACKGROUND AND PURPOSE: Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR), and exert adverse effects on mitochondrial function, although the mechanisms underlying these effects remain unclear. We used a tamoxifen-induced Hmgcr-knockout (KO) mouse model, a multi-omics approach and mitochondrial function assessments to investigate whether decreased HMGCR activity impacts key liver energy metabolism pathways. EXPERIMENTAL APPROACH: We established a new mouse strain using the Cre/loxP system, which enabled whole-body deletion of Hmgcr expression. These mice were crossed with Rosa26Cre mice and treated with tamoxifen to delete Hmgcr in all cells. We performed transcriptomic and metabolomic analyses and thus evaluated time-dependent changes in metabolic functions to identify the pathways leading to cell death in Hmgcr-KO mice. KEY RESULTS: Lack of Hmgcr expression resulted in lethality, due to acute liver damage caused by rapid disruption of mitochondrial fatty acid ß-oxidation and very high accumulation of long-chain (LC) acylcarnitines in both male and female mice. Gene expression and KO-related phenotype changes were not observed in other tissues. The progression to liver failure was driven by diminished peroxisome formation, which resulted in impaired mitochondrial and peroxisomal fatty acid metabolism, enhanced glucose utilization and whole-body hypoglycaemia. CONCLUSION AND IMPLICATIONS: Our findings suggest that HMGCR is crucial for maintaining energy metabolism balance, and its activity is necessary for functional mitochondrial ß-oxidation. Moreover, statin-induced adverse reactions might be rescued by the prevention of LC acylcarnitine accumulation.

Elevated vascular γ-butyrobetaine levels attenuate the development of high glucose-induced endothelial dysfunction.

The aim of the present study was to investigate the effects of vascular tissue levels of l-carnitine and its precursor, γ-butyrobetaine (GBB), on the development of endothelial dysfunction induced by 5 µmol/L lysophosphatidylcholine (LPC), 10 mmol/L triglycerides (TG) or a high glucose concentration (44 mmol/L). Changes in vascular tissue levels of l-carnitine and GBB were induced by administration of l-carnitine (100 mg/kg), mildronate (100 mg/kg; an inhibitor of l-carnitine synthesis) or their combination to male Wistar rats for 2 weeks. Treatment with l-carnitine elevated vascular tissue levels of l-carnitine, whereas administration of mildronate reduced l-carnitine levels and increased GBB levels. Experimental animals that received the combination of both drugs showed elevated tissue levels of GBB. The results from organ bath experiments demonstrated that increased GBB levels with preserved l-carnitine content in vascular tissues attenuated the development of endothelial dysfunction induced by high glucose. However, changes in vascular tissue l-carnitine and GBB levels had no impact on endothelial dysfunction induced by TG or LPC. The results demonstrate that increased levels of GBB with preserved l-carnitine content in vascular tissue attenuate the development of endothelial dysfunction induced by high glucose concentrations.

Knockout of Tmlhe in mice is not associated with autism spectrum disorder phenotypes or motor dysfunction despite low carnitine levels.

Deletion of exon 2 of the trimethyllysine hydroxylase epsilon (TMLHE) gene was identified in probands with autism spectrum disorder (ASD). TMLHE encodes the first enzyme in carnitine biosynthesis, N6-trimethyllysine dioxygenase (TMLD). Researchers have suggested that carnitine depletion could be important for the development of ASD and cognitive, locomotor and social dysfunctions, but previous findings have been inconclusive regarding the specific role of endogenous carnitine. We developed a mouse knockout model with constitutive TMLD enzyme inactivation that exhibited a significant decrease in the carnitine by more than 90% compared to wild-type (WT) mice. However, we did not observe any significant social, cognitive, or repetitive-behavior changes associated with ASD in the knockout mice; muscle strength and coordination were also not affected. In addition, the life expectancy of knockout mice was similar to that of WT mice. In conclusion, knockout of Tmlh in mice does not induce an ASD phenotype or motor dysfunction despite extremely low carnitine and gamma-butyrobetaine concentrations. Moreover, inactivation of TMLD does not induce a phenotype similar to previously described primary carnitine deficiency; indeed, our results showed that low levels of carnitine sustained adequate energy production, muscle function and social behavior in mice.

Decreased long-chain acylcarnitine content increases mitochondrial coupling efficiency and prevents ischemia-induced brain damage in rats.

Long-chain acylcarnitines (LCACs) are intermediates of fatty acid oxidation and are known to exert detrimental effects on mitochondria. This study aimed to test whether lowering LCAC levels with the anti-ischemia compound 4-[ethyl(dimethyl)ammonio]butanoate (methyl-GBB) protects brain mitochondrial function and improves neurological outcomes after transient middle cerebral artery occlusion (MCAO). The effects of 14 days of pretreatment with methyl-GBB (5 mg/kg, p.o.) on brain acylcarnitine (short-, long- and medium-chain) concentrations and brain mitochondrial function were evaluated in Wistar rats. Additionally, the mitochondrial respiration and reactive oxygen species (ROS) production rates were determined using ex vivo high-resolution fluorespirometry under normal conditions, in models of ischemia-reperfusion injury (reverse electron transfer and anoxia-reoxygenation) and 24 h after MCAO. MCAO model rats underwent vibrissae-evoked forelimb-placing and limb-placing tests to assess neurological function. The infarct volume was measured on day 7 after MCAO using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Treatment with methyl-GBB significantly reduced the LCAC content in brain tissue, which decreased the ROS production rate without affecting the respiration rate, indicating an increase in mitochondrial coupling. Furthermore, methyl-GBB treatment protected brain mitochondria against anoxia-reoxygenation injury. In addition, treatment with methyl-GBB significantly reduced the infarct size and improved neurological outcomes after MCAO. Increased mitochondrial coupling efficiency may be the basis for the neuroprotective effects of methyl-GBB. This study provides evidence that maintaining brain energy metabolism by lowering the levels of LCACs protects against ischemia-induced brain damage in experimental stroke models.

The Association of Circulating L-Carnitine, γ-Butyrobetaine and Trimethylamine N-Oxide Levels with Gastric Cancer.

Our study aimed to evaluate the association between gastric cancer (GC) and higher concentrations of the metabolites L-carnitine, γ-butyrobetaine (GBB) and gut microbiota-mediated trimethylamine N-oxide (TMAO) in the circulation. There is evidence suggesting that higher levels of TMAO and its precursors in blood can be indicative of either a higher risk of malignancy or indeed its presence; however, GC has not been studied in this regard until now. Our study included 83 controls without high-risk stomach lesions and 105 GC cases. Blood serum L-carnitine, GBB and TMAO levels were measured by ultra-high-performance liquid chromatography-mass spectrometry (UPLC/MS/MS). Although there were no significant differences between female control and GC groups, we found a significant difference in circulating levels of metabolites between the male control group and the male GC group, with median levels of L-carnitine reaching 30.22 (25.78-37.57) nmol/mL vs. 37.38 (32.73-42.61) nmol/mL (p < 0.001), GBB-0.79 (0.73-0.97) nmol/mL vs. 0.97 (0.78-1.16) nmol/mL (p < 0.05) and TMAO-2.49 (2.00-2.97) nmol/mL vs. 3.12 (2.08-5.83) nmol/mL (p < 0.05). Thus, our study demonstrated the association between higher blood levels of L-carnitine, GBB, TMAO and GC in males, but not in females. Furthermore, correlations of any two investigated metabolites were stronger in the GC groups of both genders in comparison to the control groups. Our findings reveal the potential role of L-carnitine, GBB and TMAO in GC and suggest metabolic differences between genders. In addition, the logistic regression analysis revealed that the only significant factor in terms of predicting whether the patient belonged to the control or to the GC group was the blood level of L-carnitine in males only. Hence, carnitine might be important as a biomarker or a risk factor for GC, especially in males.