Application of Liquid Chromatography Coupled to Mass Spectrometry for Direct Estimation of the Total Levels of Adenosine and Its Catabolites in Human Blood.

Adenosine is a multifunctional nucleoside with several roles across various levels in organisms. Beyond its intracellular involvement in cellular metabolism, extracellular adenosine potently influences both physiological and pathological processes. In relation to its blood level, adenosine impacts the cardiovascular system, such as heart beat rate and vasodilation. To exploit the adenosine levels in the blood, we employed the liquid chromatography method coupled with mass spectrometry (LC-MS). Immediately after collection, a blood sample mixed with acetonitrile solution that is either enriched with 13C-labeled adenosine or a newly generated mixture is transferred into the tubes containing the defined amount of 13C-labeled adenosine. The 13C-enriched isotopic adenosine is used as an internal standard, allowing for more accurate quantification of adenosine. This novel protocol for LC-MS-based estimation of adenosine delivers a rapid, highly sensitive, and reproducible means for quantitative estimation of total adenosine in blood. The method also allows for quantification of a few catabolites of adenosine, i.e., inosine, hypoxanthine, and xanthine. Our current setup did not allow for the detection or quantifying of uric acid, which is the final product of adenosine catabolism. This advancement provides an analytical tool that has the potential to enhance our understanding of adenosine's systemic impact and pave the way for further investigations into its intricate regulatory mechanisms.

TREATMENT OF TUBERCULOSIS IN THE 17th CENTURY BASED ON THE ORIGINAL WORK DE PHTHISI (ON TUBERCULOSIS) FROM 1679

Galen was the first who defined phthisis as lung ulceration, accompanied by coughing, persistent low-grade fevers, and body wasting. Attempts to define tuberculosis and find the cause of the disease belong to significant errors in the period of medical theories about tuberculosis (TB). Even in the 17th century, the most common causes of this disease were pulmonary ulcers, incorrect shape and position of the lungs, or menstruation. This article endeavors to elucidate the history of TB and its therapy in the 17th century on the basis of the Latin inaugural academic disputation De phthisi (On Tuberculosis) from 1679, which was first translated into Slovak in 2021. It was written by Matthaeus Palumbini, a Hungarian physician of Slovak origin born in Turiec County (Comitatus Thurociensis) in the Kingdom of Hungary. Although this dissertation is due to the anatomical, physiological, and clinical views of the Early Modern period, the ideas about the disease inhalation route as well as the fact that the disease transmission happens indirectly through the air, are close to existing knowledge. Similarly, the TB classification, the description of indications, climatic treatment, or principles of healthy lifestyle surprisingly correlate with the current medical practice. The article is supplemented by examples of the period of drug prescriptions that constituted a part of the therapy. The archival source of the original text comes from the Digitale Sammlungen der Universitätsbibliothek Erlangen-Nürnberg.

Modelling Duchenne muscular dystrophy in vitro with newly generated, blood cell-derived induced pluripotent stem cell line ORIONi003-A.

Here, we present newly derived in vitro model for modeling Duchenne muscular dystrophy. Our new cell line was derived by reprogramming of peripheral blood mononuclear cells (isolated from blood from pediatric patient) with Sendai virus encoding Yamanaka factors. Derived iPS cells are capable to differentiate in vitro into three germ layers as verified by immunocytochemistry. When differentiated in special medium, our iPSc formed spontaneously beating cardiomyocytes. As cardiomyopathy is the main clinical complication in patients with Duchenne muscular dystrophy, the cell line bearing the dystrophin gene mutation might be of interest to the research community.

Increased Sensitivity of Cough Reflex is Not the Mechanism of Cough Attributed to Laryngopharyngeal Reflux.

OBJECTIVES: In laryngopharyngeal reflux (LPR) patients acid reaches laryngopharyngeal area and stimulates/sensitizes respiratory nerve terminals mediating cough. We addressed several hypothesis: if stimulation of respiratory nerves is responsible for coughing then acidic LPR should correlate with coughing and proton pump inhibitor (PPI) treatment should reduce both LPR and coughing. If sensitization of respiratory nerves is responsible for coughing then cough sensitivity should correlate with coughing and PPI should reduce both coughing and cough sensitivity. STUDY DESIGN/METHODS: In this prospective single center study, patients with positive reflux symptom index (RSI > 13) and/or reflux finding score (RFS > 7) and ≥1 LPR episode/24 hours were enrolled. We evaluated LPR by dual channel 24-hour pH/impedance. We determined number of LPR events with pH drop at levels 6.0, 5.5, 5.0, 4.5, and 4.0. Cough reflex sensitivity was determined as lowest capsaicin concentration causing at least 2/5 coughs (C2/C5) by single breath capsaicin inhalation challenge. For statistical analysis C2/C5 values were -log transformed. Troublesome coughing was evaluated on the scale 0-5. RESULTS: We enrolled 27 LPR patients. The number of LPR events with pH 6.0, 5.5, 5.0, 4.5, and 4.0 was 14[8-23],4[2-6],1[1-3],1[0-2] and 0[0-1], respectively. There was no correlation between number of LPR episodes at any pH level and coughing (Pearson range -0.34 to 0.21, P = NS). There was no correlation between cough reflex sensitivity C2/C5 and coughing (R = -0.29 to 0.34, P = NS). Of patients that completed PPI treatment, 11 had RSI normalized (18.36 ± 2.75 vs. 7 ± 1.35, P < 0.01). There was no change in cough reflex sensitivity in PPI-responders. C2 threshold was 1.41 ± 0.19 vs. 1.2 ± 0.19 (P = 0.11) before and after PPI. CONCLUSIONS: No correlation between cough sensitivity and coughing and no change in cough sensitivity despite improvement of coughing by PPI argue that an increased cough reflex sensitivity is not mechanism of cough in LPR. We identified no simple relationship between LPR and coughing suggesting that this relationship is more complex.

Comparison of the methods for isolation and detection of SARS-CoV-2 RNA in municipal wastewater.

Introduction: Coronavirus SARS-CoV-2 is a causative agent responsible for the current global pandemic situation known as COVID-19. Clinical manifestations of COVID-19 include a wide range of symptoms from mild (i.e., cough, fever, dyspnea) to severe pneumonia-like respiratory symptoms. SARS-CoV-2 has been demonstrated to be detectable in the stool of COVID-19 patients. Waste-based epidemiology (WBE) has been shown as a promising approach for early detection and monitoring of SARS-CoV-2 in the local population performed via collection, isolation, and detection of viral pathogens from environmental sources. Methods: In order to select the optimal protocol for monitoring the COVID-19 epidemiological situation in region Turiec, Slovakia, we (1) compared methods for SARS-CoV-2 separation and isolation, including virus precipitation by polyethylene glycol (PEG), virus purification via ultrafiltration (Vivaspin®) and subsequent isolation by NucleoSpin RNA Virus kit (Macherey-Nagel), and direct isolation from wastewater (Zymo Environ Water RNA Kit); (2) evaluated the impact of water freezing on SARS- CoV-2 separation, isolation, and detection; (3) evaluated the role of wastewater filtration on virus stability; and (4) determined appropriate methods including reverse transcription-droplet digital PCR (RT-ddPCR) and real-time quantitative polymerase chain reaction (RT-qPCR) (targeting the same genes, i.e., RdRp and gene E) for quantitative detection of SARS-CoV-2 in wastewater samples. Results: (1) Usage of Zymo Environ Water RNA Kit provided superior quality of isolated RNA in comparison with both ultracentrifugation and PEG precipitation. (2) Freezing of wastewater samples significantly reduces the RNA yield. (3) Filtering is counterproductive when Zymo Environ Water RNA Kit is used. (4) According to the specificity and sensitivity, the RT-ddPCR outperforms RT-qPCR. Discussion: The results of our study suggest that WBE is a valuable early warning alert and represents a non-invasive approach to monitor viral pathogens, thus protects public health on a regional and national level. In addition, we have shown that the sensitivity of testing the samples with a nearer detection limit can be improved by selecting the appropriate combination of enrichment, isolation, and detection methods.

Flavonoids attenuate cancer metabolism by modulating Lipid metabolism, amino acids, ketone bodies and redox state mediated by Nrf2.

Metabolic reprogramming of cancer cells is a common hallmark of malignant transformation. The preference for aerobic glycolysis over oxidative phosphorylation in tumors is a well-studied phenomenon known as the Warburg effect. Importantly, metabolic transformation of cancer cells also involves alterations in signaling cascades contributing to lipid metabolism, amino acid flux and synthesis, and utilization of ketone bodies. Also, redox regulation interacts with metabolic reprogramming during malignant transformation. Flavonoids, widely distributed phytochemicals in plants, exert various beneficial effects on human health through modulating molecular cascades altered in the pathological cancer phenotype. Recent evidence has identified numerous flavonoids as modulators of critical components of cancer metabolism and associated pathways interacting with metabolic cascades such as redox balance. Flavonoids affect lipid metabolism by regulating fatty acid synthase, redox balance by modulating nuclear factor-erythroid factor 2-related factor 2 (Nrf2) activity, or amino acid flux and synthesis by phosphoglycerate mutase 1. Here, we discuss recent preclinical evidence evaluating the impact of flavonoids on cancer metabolism, focusing on lipid and amino acid metabolic cascades, redox balance, and ketone bodies.

Correlation between α-synuclein and fatty acid composition in jejunum of rotenone-treated mice is dependent on acyl chain length.

Events associated with the progression of Parkinson´s disease (PD) are closely related to biomembrane dysfunction. The specific role of membrane composition in the conformational stability of alpha synuclein (αS) has already been well documented. Administration of rotenone is one of the best strategies to initiate PD phenotype in animal models. In the present study, daily exposure (14 weeks) of orally administered rotenone (10 mg/kg) was employed in a mouse model. The mitochondrial complex I inhibition resulted in elevated level of αS in whole tissue homogenate of mouse jejunum. In addition, we identified a strong intra-individual correlation between αS level and the specific esterified fatty acids. The observed correlation depends mainly on the acyl chain length. Based on the obtained results, it is suggested that there is a high potential to manipulate fatty acid homeostasis in modulating αS based pathogenesis of PD, at least in experimental conditions.

In vitro modeling of amyotrophic lateral sclerosis with induced pluripotent stem cell technology-derived cell line ORIONi002-A.

We present here a new iPS cell line for modeling sporadic form of ALS. Cell line was generated by reprogramming skin fibroblasts isolated with explant culture technology from skin biopsy, donated by ALS patient. For reprogramming, polycistronic self-replicating RNA vector was used and derived iPS cells were characterized by immunocytochemistry and FACS (pluripotent factors expression), karyotyping, STR fingerprinting analysis and in vitro differentiation assay. New cell line showed normal (46, XY) karyotype and differentiated in vitro into cells from three germ layers. STR analysis proved the origin and originality of the cell line.

Changes in Cough Reflex Sensitivity in Children After Removal of Hypertrophied Adenoid Tissue.

Purpose: The co-occurrence of adenoids and chronic cough is common in children. The goal of this research was to specify changes in cough reflex sensitivity as a result of adenoid tissue removal. Patients and Methods: The sample group consisted of 17 children (six boys and 11 girls, aged 4-12 years, mean age 6.24 years), all of them possessing symptoms of chronic cough and adenoids, confirmed by nasal fiberoptic endoscopy. This sample group underwent cough reflex sensitivity assessment, which took place both prior to and after endoscopic adenoidectomy. The definition of the cough reflex sensitivity is the lowest capsaicin concentration that caused two (C2) or five (C5) coughs. Capsaicin aerosol in ascending concentrations (from 0.61 to 1250 µmol/L) was inhaled by a single-breath method (KoKo DigiDoser), with the addition of an inspiratory flow regulator valve (RIFR). Results: Concentrations of capsaicin causing two (C2) and five coughs (C5) were reported. Cough sensitivity (geometric mean with 95% CI) for C2 was 31.86 (12.98-78.18) µmol/L preoperatively and 11.97 (6.16-23.26) µmol/L postoperatively (P=0.064). Cough sensitivity for C5 was 234.91 (97.19-567.77) µmol/L preoperatively and 69.13 (29.08-164.35) µmol/L postoperatively (P=0.022). The children's pulmonary function was within the normal range. Conclusion: In our study, adenoidectomy significantly increased cough reflex sensitivity in non-atopic children suffering from chronic cough.

Cough reflex sensitivity does not correlate with the esophageal sensitivity to acid in patients with gastroesophageal reflux disease.

The sensitization of cough reflex observed in patients with gastroesophageal reflux disease (GERD) is attributed to activation of vagal C-fibers innervating the esophagus by acid, while the heartburn in GERD is mediated by esophageal acid sensitive C-fibers derived from (dorsal root ganglia) DRG. Here we explored the relationship between cough reflex sensitivity (CRS) and esophageal sensitivity to acid. We evaluated CRS to capsaicin inhalation and esophageal sensitivity to acid (intensity of heartburn evoked by esophageal infusions of acid pH = 3, 2 and 1) in patients with GERD and chronic heartburn before and 3 months after proton pump inhibitor (PPI) treatment. There was no correlation between CRS and esophageal sensitivity to acid at any pH tested. PPI treatment substantially reduced esophageal sensitivity to acid but did not affect CRS. We conclude that a simple direct relationship between CRS and esophageal sensitivity to acid is unlikely. The results indicate that spinal and vagal afferent pathways from the esophagus are probably influenced separately in subjects with GERD.

The effect of adenoidectomy on transnasal airflow in children with hypertrophy of adenoid tissue.

BACKGROUND: The aim of this study was to clarify changes of transnasal airflow resulting from adenoidectomy and to assess the effect of surgery depending on adenoid hypertrophy (AH) obstruction grade. MATERIALS AND METHODS: Altogether fifty children having symptoms of nasal obstruction and adenoids were submitted to a rhinomanometric assessment before and after adenoidectomy. At the same time, using the nasal fiberoptic endoscopy, the grade of AH obstruction was determined, according to which the children were divided into four classes. We assessed the change of total transnasal inspiratory airflow and total nasal resistance due to adenoidectomy. RESULTS: Values of transnasal airflow and nasal resistance measured in the study group of fifty children were preoperatively 262 mL/s and 0.565 kPa/L/s; postoperatively 288 mL/s and 0.52 kPa/L/s. We have noticed statistically significant increase of the airflow (P = 0.015); however, decrease of the resistance (P = 0.054) was not significant. In the group of children suffering from the 1st to 2nd grade (29 children) preoperatively measured values presented 280 mL/s and 0.52 kPa/L/s; postoperatively, 276 mL/s and 0.54 kPa/L/s; change of the airflow (P = 0.634) and resistance (P = 0.829) was not significant. In the study group having the 3rd and 4th grade (21 children), the values indicated preoperatively 240 mL/s and 0.62 kPa/L/s; postoperatively, 340 mL/s and 0.44 kPa/L/s; there were significant airflow increase (P = 0.012) and resistance decrease (P = 0.033). CONCLUSIONS: Adenoidectomy significantly increased the airflow; however, we observed the different effect in the group of children with the 1st and 2nd grade compared to the group with the 3rd and 4th grade. A significant increase of the airflow and decrease of the resistance were present only in the group with the 3rd and 4th grade; therefore, the significant reduction of nasal obstruction symptoms might be expected only in this group of patients.

Mechanisms of the cough associated with rhinosinusitis.

The diseases of the nose and paranasal sinuses (rhinosinusitis) often in combination with asthma and gastroesophageal reflux are common causes of chronic cough in patients with normal chest radiograph. The relationships between rhinosinusitis and cough are incompletely understood. We investigated modulation of the cough reflex by the inputs from the nose. We demonstrate that the cough reflex is sensitized by the intranasal administration of sensory nerve activators in animal models and in humans. Cough reflex is also sensitized in the guinea pig model of allergic nasal inflammation and in patients with allergic rhinitis. In patients with allergic rhinitis the cough sensitization is augmented during the allergen season. We conclude that the cough reflex can be sensitized from the nose. Our data indicate that this sensitization is mediated by nasal sensory nerves. We speculate that by inducing the cough reflex sensitization rhinosinusitis contributes to chronic cough. If combined with environmental or endogenous cough triggers, the cough reflex sensitization is predicted to cause excessive coughing. The potential endogenous cough triggers may be associated with rhinosinusitis (postnasal drip, aspiration of nasal secrets) or secondary to a coexistent disease such as asthma or gastroesophageal reflux.