Michal Jan Markuszewski

Background: Excessive alcohol consumption constitutes a serious cause of death worldwide. Fatty acid ethyl esters, as metabolites of the non-oxidative elimination pathway of ethanol, have been recognized as mediators of alcohol-induced organ damage. These metabolites serve as potential biomarkers for the assessment of ethanol intake and might be also used in post-mortem studies. Methods: In this study, the development and optimization of a simple, fast, precise, accurate, and cost-effective method with the use of gas chromatography coupled with tandem mass spectrometry for quantitative analysis of six fatty acid ethyl esters, namely ethyl laurate, myristate, palmitate, linoleate, oleate, and stearate, were conducted. Results: The optimized method was fully validated according to ICH guidelines. Additionally, identification of critical method parameters was possible by using the quality by design approach. By carrying out analyses according to the Plackett–Burman plan (design of experiments methodology), the robustness of the analytical method developed was confirmed for four (ethyl palmitate, linoleate, oleate, and stearate) ethyl esters. In the case of ethyl myristate, the variable significantly affecting the results appeared to be the temperature of solvent evaporation after the deproteinization step. Conclusions: Biochemical interpretation of the obtained results with available medical records suggests that plasma concentrations of selected fatty acid ethyl esters are valuable indicators of pre-mortem alcohol consumption and may be one of the key factors helpful in determining the cause and mechanism of death.

Background: Pulmonary arterial hypertension (PAH) is a rare and severe condition characterized by increased pulmonary arterial pressure and vascular resistance. Women are more susceptible to PAH yet have higher survival rates than men, a phenomenon called the “estrogen paradox”. This study aims to investigate the sex-based differences in PAH using plasma untargeted metabolomics. Methods: Plasma samples were collected from 43 PAH patients and 37 healthy controls. The samples were analyzed using two complementary analytical techniques: gas chromatography–mass spectrometry (GC-QqQ/MS) and liquid chromatography–mass spectrometry (LC-Q-ToF/MS). The metabolic differences between male and female PAH patients and controls were identified using multivariate statistical analyses. Results: Our results show changes in the lipid, fatty acid, and amino acid metabolism in both sexes. Women presented additional changes in the carbohydrate, bile acid, and nucleotide metabolism. The metabolites affected by PAH in women included decreased threonine, tryptophan, and lipid intermediates and elevated bile acids. Men were found to have additional changes in the heme catabolism, cholesterol synthesis, and lipoxygenase pathways. The metabolites affected by PAH in men included decreased branched-chain amino acids and increased bilirubin, phospholipids, and oxidized fatty acids. Conclusions: The gender differences observed in the development of PAH are likely multifactorial. While estrogens and potentially other sex hormones have been implicated in modulating relevant biological pathways, their exact role in disease progression and pathogenesis remains to be fully elucidated. The specific metabolic changes in women and men point to distinct disease mechanisms, potentially explaining the differences in prevalence, prognosis, and treatment response of patients with PAH. The obtained results should be validated with the use of targeted quantitative analyses and larger numbers of patients.

In the absence of fully effective therapies and preventive strategies against the development of urosepsis, a deeper understanding of the virulence mechanisms of Uropathogenic Escherichia coli (UPEC) strains is needed. UPEC strains employ a wide range of virulence factors (VFs) to persist in the urinary tract and bloodstream. UPEC strains were isolated from patients with sepsis and a control group without sepsis. PCR was used to detect 36 genes encoding various groups of virulence and fitness factors. Profiling of both intracellular and extracellular bacterial proteins was also included in our approach. Bacterial metabolites were identified and quantified using GC-MS and LC-MS techniques. The UpaG autotransporter, a trimeric E. coli AT adhesin, was significantly more prevalent in urosepsis strains (p = 0.00001). Iron uptake via aerobactin and the Iha protein also appeared to be predictive of urosepsis (p = 0.03 and p = 0.002, respectively). While some studies suggest an association between S fimbriae and the risk of urosepsis, we observed no such correlation (p = 0.0001). Proteomic and metabolomic analyses indicated that elevated levels of bacterial citrate, malate, coenzyme Q10, pectinesterase (YbhC), and glutamate transport proteins, as well as the regulators PhoP two-component system, CpxR two-component system, Nitrate/nitrite response regulator protein NarL, and the Ferrienterobactin receptor FepA, may play a role in sepsis. These genetic biomarkers, proteins, and metabolites derived from UPEC could potentially serve as indicators for assessing the risk of developing sepsis.

Background/Objectives: Atrial fibrillation (AF) is the most frequent arrhythmia in the adult population associated with a high rate of severe consequences leading to significant morbidity and mortality worldwide. Therefore, its prompt recognition is of high clinical importance. AF detection often remains challenging due to unspecific symptoms and a lack of reliable biomarkers for its prediction. Herein, novel bioanalytical methodologies, such as metabolomics, offer new opportunities for a better understanding of the underlying pathological mechanisms of cardiovascular diseases, including AF. The metabolome, considered a complete set of small molecules present in the organism, directly reflects the current phenotype of the studied system and is highly sensitive to any changes, including arrhythmia’s onset. A growing body of evidence suggests that metabolite profiling has prognostic value in AF prediction, highlighting its potential role not only in early diagnosis but also in guiding therapeutic interventions. By identifying specific metabolites as a disease biomarker or recognising particular metabolomic pathways involved in the AF pathomechanisms, metabolomics could be of great clinical value for further clinical decision-making, risk stratification, and an individual personalised approach. The presented narrative review aims to summarise the current state of knowledge on metabolomics in AF with a special emphasis on its implications for clinical practice and personalised medicine.

Graphene and graphene-based materials have attracted growing interest for potential applications in medicine because of their good biocompatibility, cargo capability and possible surface functionalizations. In parallel, prototypic graphene-based devices have been developed to diagnose, imaging and track tumor growth in cancer patients. There is a growing number of reports on the use of graphene and its functionalized derivatives in the design of innovative drugs delivery systems, photothermal and photodynamic cancer therapy, and as a platform to combine multiple therapies. The aim of this review is to introduce the latest scientific achievements in the field of innovative composite graphene materials as potentially applied in cancer therapy. The “Technology and Innovation Roadmap” published in the Graphene Flagship indicates, that the first anti-cancer drugs using graphene and graphene-derived materials will have appeared on the market by 2030. However, it is necessary to broaden understanding of graphene-based material interactions with cellular metabolism and signaling at the functional level, as well as toxicity. The main aspects of further research should elucidate how treatment methods (e.g., photothermal therapy, photodynamic therapy, combination therapy) and the physicochemical properties of graphene materials influence their ability to modulate autophagy and kill cancer cells. Interestingly, recent scientific reports also prove that graphene nanocomposites modulate cancer cell death by inducing precise autophagy dysfunctions caused by lysosome damage. It turns out as well that developing photothermal oncological treatments, it should be taken into account that near-infrared-II radiation (1000– 1500 nm) is a better option than NIR-I (750– 1000 nm) because it can penetrate deeper into tissues due to less scattering at longer wavelengths radiation.

Among the currently available commercial eye drops with cyclosporine A (Cs) there is a lack of long-acting dosage forms and products with a concentration of the drug substance higher than 0.1%, although Cs is widely used in ophthalmology. The aim of the research was to conduct the microscopic and biopharmaceutical evaluation of two formulations, an emulsion (EM) and a self-emulsifying oil (SEO), both with 0.5% of Cs, proposed for use in eye drops, and the comparison of both. SEO eye drops with Cs or any other drug substance are currently not available as marketed products, and the highest concentration of Cs in the ocular emulsion is only 0.1%. The microscopic evaluation of the emulsion and the SEO after emulsification with water was carried out using a high-resolution digital microscopy. The properties of both preparations were compared using the high dynamic range function or optical shadow effect mode. Images in the 3D composition mode were also recorded. The in vivo study of the Cs formulations was performed on male albino rabbits. The eye tolerance of the preparations was assessed using the ocular irritation test, which is a modified Draize test. Placebo carriers (without the drug substance) were also subjected to irritation testing. The concentration of Cs in the tissues (cornea and conjunctiva) and fluids (tear fluid and aqueous humor) of the rabbit eye was determined after multiple instillations of Cs–EM or Cs–SEO. The tested preparations were compared using the digital microscopy technique, which highlights the features of the formulations and eliminates the risk of unnoticeable properties that are difficult to observe in classical optical microscopy. Both tested Cs-loaded formulations are classified as practically non-irritating. There were also no significant differences when testing the placebo carriers. After a topical administration, Cs was widely distributed in all tissues (e.g., in cornea 1.3 ng/mg and 1.0 ng/mg) and fluids of the eye (e.g., in tear fluid 11.6 µg/mL and 4.3 µg/mL), after the administration of Cs–SEO and Cs–EM, respectively. The obtained results allow us to recognize both tested formulations, the emulsion and the self-emulsifying oil with 0.5% Cs content, as carriers safe for ophthalmic use and effective in delivering the drug substance to the structures of the eye.