Lipids from microorganisms, and especially lipids from Archaea, are used as taxonomic markers. Unfortunately, knowledge is very limited due to the uncultivability of most Archaea, which greatly reduces the importance of the diversity of lipids and their ecological role. One possible solution is to use lipidomic analysis. Six radioactive sources were investigated, two of which are surface (Wettinquelle and Radonka) and four deep from the Svornost mine (Agricola, Behounek, C1, and Curie). A total of 15 core lipids and 82 intact polar lipids were identified from the membranes of microorganisms in six radioactive springs. Using shotgun lipidomics, typical Archaea lipids were identified in spring water, namely dialkyl glycerol tetraethers, archaeol, hydroxyarchaeol and dihydroxyarchaeol. Diverse groups of polar heads were formed in archaeal IPLs, whose polar heads are formed mainly by hexose, deoxyhexose, and phosphoglycerol. The analysis was performed using shotgun lipidomics and the structure of all molecular species was confirmed by tandem mass spectrometry. After acid hydrolysis, a mixture of polar compounds was obtained from the polar head. Further analysis by GC-MS confirmed that the carbohydrates were glucose and rhamnose. Analysis by HPLC-MS of diastereoisomers of 2-(polyhydroxyalkyl)-3-(O-tolylthiocarbamoyl)thiazolidine-4(R)-carboxylates revealed that both L-rhamnose and D-glucose are present in spring samples only in varying amounts. The glycoside composition depends on the type of spring, that is, Wettinquelle and Radonka springs are basically shallow groundwater, while the samples from the Svornost mine are deep groundwater and do not contain glycosides with rhamnose. This method enables quick screening for characteristic Archaea lipids, allowing decisions on whether to pursue further analyses, such as metagenomic analysis, to directly confirm the presence of Archaea.
The present study has undertaken the isolation of marine yeasts from mangrove sediment samples and their ability to produce alkaline protease enzymes. A total of 14 yeast isolates were recovered on yeast-malt agar (YMA) and yeast extract peptone dextrose (YEPD) agar medium. After screening for proteolytic activity on skim milk agar, marine yeast isolate, AKB-1 exhibited a hydrolysis zone of 18 mm. Optimal conditions for the enzyme production from yeast isolate AKB-1 were at 30 °C, pH 8, fructose as carbon source, potassium nitrate as nitrogen source, and 25% saline concentration. Under the optimal conditions, the protease enzyme activity of the isolate AKB-1 was observed to be 978 IU/mL. The structural and functional analysis was carried out through FTIR and HPLC analysis for the extracted protease enzyme. Furthermore, the enzyme produced was partially purified by solvent extraction using ethyl acetate and ammonium sulfate precipitation (3.4-fold) followed by dialysis (56.8-fold). The molecular weight of the purified enzyme was observed to be around 60 kDa using SDS-PAGE. The extracted protein showed good antibacterial activity against six different clinical bacterial pathogens and the highest against Bacillus cereus (16 ± 0.5 mm). The extracted protease enzyme was revealed to remove blood stains from cloth within 20 min of application similar to the commercial detergent. The marine yeast isolate was further identified as Candida orthopsilosis AKB-1 (Accession number KY348766) through 18S rRNA sequencing, and a phylogenetic tree was generated.
- MeSH
- Anti-Bacterial Agents pharmacology metabolism chemistry isolation & purification MeSH
- Bacillus cereus drug effects MeSH
- Bacterial Proteins * chemistry pharmacology metabolism isolation & purification MeSH
- Candida * enzymology isolation & purification genetics classification MeSH
- Endopeptidases * chemistry metabolism isolation & purification pharmacology MeSH
- Phylogeny MeSH
- Geologic Sediments microbiology MeSH
- Hydrogen-Ion Concentration MeSH
- Culture Media chemistry MeSH
- Microbial Sensitivity Tests MeSH
- Molecular Weight MeSH
- Enzyme Stability MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
Among carotenoids, ꞵ-carotene has the highest biological activity and is found as an all-trans isomer in many biological systems. Blakeslea trispora is a microorganism that is of interest to industries for the commercial production of ꞵ-carotene. This study investigated the effect of different bacteria on carotenogenesis in B. trispora. The B. trispora bisexual mold was cultured in a production medium, and different bacterial cells were added to it after 24 h. Then, the culture conditions and the culture medium were optimized in the presence of the selected bacteria using the experimental design. The percentage of carotenoids obtained from the mixed culture was determined using high-performance liquid chromatography (HPLC). Results showed that Kocuria rhizophila had the greatest effect on increasing the production of carotenoids in B. trispora. The highest content of carotenoids obtained during optimization was 770 ± 7.5 mg/L, a 6.8-fold increase compared to the control. HPLC analysis of carotenoids indicated the presence of two main peaks, ꞵ-carotene and γ-carotene, in which the primary carotenoid was ꞵ-carotene followed by γ-carotene with a lower content. Therefore, due to the importance of ꞵ-carotene in industry, the use of biostimulants is one of the appropriate strategies to increase the production of this pigment in industry.
In this study, simple oil-in-water emulsions (O/W) and multiple O/W/O emulsions were employed as carriers for a curcumin delivery system. The stability of emulsions was evaluated using DSC (differential scanning calorimetry), accompanied by particle size measurement by DLS (dynamic light scattering) and rheological analysis. The amount of freezable water (Wfs) in O/W emulsion was determined to be 80.4%, while that in O/W/O emulsion was 23.7%. Multiple emulsions had a more complex structure than simple emulsions, being characterized by higher stability with predominant loss modulus over storage modulus (G" > G'). The mean surface diameter for O/W emulsion was 198.7 ± 9.8 nm, being approximately two times lower than that for multiple emulsions. Curcumin in vitro digestibility was observed for both emulsions and, additionally, the digestibility of fresh and dried curcuma root powders was investigated. Multiple emulsions were found to be a superior matrix for curcumin delivery, with higher stability and emulsion digestibility of 50.6% for the stomach and small intestine. In vitro digestion of dried curcuma powders and curcuma root samples was monitored by HPLC (high-performance liquid chromatography). The DMD (dry matter digestibility) for dried curcuma powders ranged between 52.9% to 78.8%, and for fresh curcuma (KF) was 95.5%.
Three types of solid waste are produced during beer fermentation: spent grain, hot trub, and residual yeast. While the first is used as livestock feed, the seconds has not yet found any real reapplication in the field of circular economy. The aim of this work is to study and characterize these two brewing wastes, i.e., hot trub and residual yeast, to evaluate their potential reuse in the agricultural field. Samples from top-fermented and bottom-fermented beers were chemically investigated. Initially, the safety was assessed via multi-detection analysis of 57 mycotoxins, and all samples were deemed safe. Subsequently, the chemical and elemental composition was examined via ICP-MS and microanalysis, along with phenolic compounds and antioxidant activity via HPLC and spectrophotometric determinations, to achieve a thorough characterization of these waste samples. The C/N ratio of residual yeast from top-fermented beer and hot trub of the bottom-fermented one were near the optimal one (10:1). This research marks an initial step towards repurposing brewery waste materials as fertilizers. The subsequent steps will involve the formulation and field trials.
- Publication type
- Journal Article MeSH
OBJECTIVE: The aim of the study was the assessment of adherence to antiretroviral (ARV) treatment in a population of people living with HIV (PWH), improving the awareness of PWH, drawing attention to the risk of developing HIV drug resistance and subsequent treatment failure. METHODS: The basic cohort consisted of PWH followed up long-term at the HIV centre of the University Hospital Pilsen. Adherence to treatment was assessed by ARV levels. Nucleoside analogs were determined in urine by high pressure liquid chromatography (HPLC), in relation to clinical data, viral load (HIV RNA), and absolute CD4 and CD8 T cell counts. To assess mental and physical state of the patients, a modified SF-36 questionnaire was used to measure social relationships, education and ability to relax. RESULTS: From a group of 131 PWH, 18 (13.7%) with zero levels and 113 (86.3%) with any detectable ARV levels were followed for 6-12 months. A statistically significant lower viral load was demonstrated in patients who adhered to the treatment at the time of the test as indicated by ARV levels in the urine. CD4 T lymphocyte values in adherent patients were, as expected, statistically significantly higher. A significant difference for CD8 T lymphocyte was not demonstrated. A survey assessed subjective factors influencing the degree of adherence. PWH consider important: quality care enabling trust, low risk of developing opportunistic infections, self-sufficiency, quality of sleep, managing leisure activities, and good family relationships. Quality of life evaluation and satisfaction in the monitored areas were similar in both groups of PWH. CONCLUSIONS: Non-adherence leads to deterioration of CD4 and viral load levels and may be the cause of the development of HIV drug resistance and treatment failure on the part of the patient. PWH with zero or low urinary nucleoside levels were repeatedly instructed about the need for regular and sustained medication use. Regular checks with a laboratory examination service are needed to detect early emergence of resistance and side effects of the treatment, which are initially only detectable in the laboratory.
- MeSH
- Medication Adherence * psychology MeSH
- Adult MeSH
- HIV Infections * drug therapy psychology MeSH
- Cohort Studies MeSH
- Quality of Life * MeSH
- Anti-HIV Agents * therapeutic use urine MeSH
- Middle Aged MeSH
- Humans MeSH
- CD4 Lymphocyte Count MeSH
- Viral Load MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Multidimensional chromatography coupled to tandem mass spectrometry (MS/MS), including simple sample preparation with protein precipitation, anion conversion with ammonium hydroxide, and solid-phase extraction using mixed-mode anion exchange in a 96-well plate format, has been validated for rapid simultaneous analysis of human insulin and its six analogs (lispro, glulisine, glargine, degludec, detemir, and aspart) in human plasma. This method is critical for clinical diagnostics, forensic investigations, and anti-doping efforts due to the widespread use of these substances. In the present study, improved chromatographic resolution was achieved using a first-dimension trap-and-elute configuration with an XBridge C18 (2.1 × 20 mm, 3.5 μm) trap column combined with second dimension separation on a Cortecs Ultra-High-Performance Liquid Chromatography (UHPLC) C18+ (2.1 × 100 mm, 1.6 μm) analytical column implemented within a two-dimensional-LC-MS/MS system. The total chromatographic run time was 11 min. This setup increases both the resolution and sensitivity of the method. A mobile phase consisting of 0.8% formic acid (FA) in water and 0.7% FA in acetonitrile was used for gradient elution. Bovine insulin was used as the internal standard. MS detection was performed in positive electrospray ionization mode, and the ion suppression due to matrix effects was evaluated. Validation criteria included linearity, precision, accuracy, recovery, lower limit of quantitation, matrix effect, and stability tests with and without protease inhibitor cocktail under different conditions (short-term stability, long-term stability, and freeze-thaw stability). The concentration range for all insulins was 50-15 000 pg/mL, with limits of quantification below the therapeutic reference range for all analytes. Intra-run precision ranged from 1.1% to 5.7%, inter-run precision from 0.7% to 5.9%, and overall recovery from 96.9% to 114.3%. The validated method has been implemented successfully by the Department of Forensic Medicine at our hospital for the investigation of unexplained deaths.
Epilepsy, affecting over 50 million people globally, presents a significant neurological challenge. Effective prevention of epileptic seizures relies on proper administration and monitoring of Anti-Seizure Medication (ASMs). Therapeutic Drug Monitoring (TDM) ensures optimal dosage adjustment, minimizing adverse effects and potential drug interactions. While traditional venous blood collection for TDM may be stressful, emerging alternative sampling methods, particularly Dried Blood Spot (DBS) or oral fluid offer less invasive way of sampling. This study aimed to develop and validate an analytical method for the determination of lamotrigine in such alternative samples. The sample, either DBS or oral fluid, was subjected to extraction, evaporation, and reconstitution in 15 % acetonitrile containing 0.1 % formic acid. A Kinetex C18 Polar column was used for liquid chromatographic separation and MS in ESI+ mode was used for detection and quantitation of lamotrigine using an isotopically labelled internal standard according to EMA guidelines. The calibration range of the developed method enables the determination of lamotrigine in the concentration range of 1-30 μg/mL in DBS and 0.5-20 μg/mL in oral fluid. Oral fluid and DBS samples from patients treated with lamotrigine analysed by the developed method were compared to plasma concentrations measured by the hospital's accredited laboratory. Preliminary results indicate a promising potential for these alternative matrices in clinical TDM applications. By offering a less invasive sampling approach, this method improves the accessibility and safety of pharmacotherapy for epilepsy patients. The results of this study lay the foundation for further clinical applications by implementing alternative matrix TDM, which may significantly advance personalized care in epilepsy management.
- MeSH
- Anticonvulsants * analysis blood MeSH
- Chromatography, Liquid methods MeSH
- Epilepsy drug therapy MeSH
- Calibration MeSH
- Liquid Chromatography-Mass Spectrometry MeSH
- Lamotrigine * analysis blood MeSH
- Humans MeSH
- Limit of Detection MeSH
- Drug Monitoring * methods MeSH
- Reproducibility of Results MeSH
- Saliva * chemistry MeSH
- Tandem Mass Spectrometry methods MeSH
- Dried Blood Spot Testing * methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Validation Study MeSH
The precise and unambiguous detection and quantification of internal RNA modifications represents a critical step for understanding their physiological functions. The methods of direct RNA sequencing are quickly developing allowing for the precise location of internal RNA marks. This detection is, however, not quantitative and still presents detection limits. One of the biggest remaining challenges in the field is still the detection and quantification of m6A, m6Am, inosine, and m1A modifications of adenosine. The second intriguing and timely question remaining to be addressed is the extent to which individual marks are coregulated or potentially can affect each other. Here, we present a methodological approach to detect and quantify several key mRNA modifications in human total RNA and in mRNA, which is difficult to purify away from contaminating tRNA. We show that the adenosine demethylase FTO primarily targets m6Am marks in noncoding RNAs in HEK293T cells. Surprisingly, we observe little effect of FTO or ALKBH5 depletion on the m6A mRNA levels. Interestingly, the upregulation of ALKBH5 is accompanied by an increase in inosine level in overall mRNA.
- MeSH
- Adenosine * analogs & derivatives metabolism genetics analysis MeSH
- AlkB Homolog 5, RNA Demethylase * metabolism genetics MeSH
- Chromatography, Liquid methods MeSH
- Alpha-Ketoglutarate-Dependent Dioxygenase FTO * metabolism genetics MeSH
- HEK293 Cells MeSH
- Inosine * metabolism genetics MeSH
- Liquid Chromatography-Mass Spectrometry MeSH
- Humans MeSH
- RNA, Messenger * genetics metabolism MeSH
- RNA Processing, Post-Transcriptional MeSH
- Tandem Mass Spectrometry * methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Boswellia resin is an exudate from the cut bark of Boswellia trees. The main constituents of pharmacological interest are boswellic acids (pentacyclic triterpenoids), namely α-boswellic acid, β-boswellic acid, 3-O-acetyl-α-boswellic acid, 3-O-acetyl-β-boswellic acid, 11-keto-β-boswellic acid, and 3-O-acetyl-11-keto-β-boswellic acid. Nowadays, dietary supplements with Boswellia serrata extract are used in the treatment of inflammatory joint diseases. Additionally, the constituents of Boswellia resin have shown potential for the treatment of other chronic inflammatory diseases and various types of cancer. Separation methods including ultra/high-performance liquid chromatography, gas chromatography, thin layer chromatography, supercritical fluid chromatography, and capillary electrochromatography coupled with UV or MS detection have been used for the determination of boswellic acids in various matrices (mostly plant material and biological samples). This review aims to provide a comprehensive summary of these separation methods, offering a critical discussion of their strengths and limitations in the analysis of boswellic acids. The knowledge of various separation methods plays a pivotal role in the quality control of herbal dietary supplements and the monitoring of the metabolism and pharmacokinetics of their constituents. The approaches based on metabolomics and network pharmacology represent new ways of fingerprinting secondary metabolites in Boswellia resin increasing the comprehensiveness of the output of these methods resulting in safer dietary supplements.
- MeSH
- Boswellia chemistry MeSH
- Humans MeSH
- Plant Extracts chemistry MeSH
- Triterpenes * analysis isolation & purification MeSH
- Chromatography, High Pressure Liquid MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
