INTRODUCTION: Ziziphora clinopodioides subsp. bungeana (Juz.) Rech.f. is used in traditional medicine for various purposes. Previous phytochemical studies focused on phenolic compounds, but triterpenoids were almost overlooked. OBJECTIVE: The study focused on the isolation of compounds with dual antidiabetic activity from the aerial parts of Z. clinopodioides subsp. bungeana. MATERIALS AND METHODS: Separation of CHCl3-soluble fraction by silica gel column chromatography using different mobile phases and purification of compounds by semi-preparative HPLC or preparative TLC. The structures of pure compounds were elucidated by 1D and 2D NMR experiments along with HRMS. Compound 1 was additionally identified by the single crystal X-ray diffraction method. α-Glucosidase inhibitory assay and GLUT4 expression and translocation in C2C12 myotubes were conducted to evaluate antidiabetic potential of isolated compounds. RESULTS: This phytochemical study led to the isolation of 20 compounds, including a unique monoterpene diperoxy dimer (1). Compounds 7 and 9-11 displayed more potent α-glucosidase inhibitory activity (IC50 45.3-135.3 μM) than acarbose used as a positive control (IC50 264.7 μM), while only pomolic acid (5) increased GLUT4 translocation in C2C12 myotubes in a significant manner. CONCLUSION: Extensive chromatographic separation led to the isolation and identification of a unique monoterpene diperoxy dimer (1) from aerial parts of Z. clinopodioides subsp. bungeana. Some triterpenes inhibited α-glucosidase, another increased GLUT4 translocation. Although none of the isolated compounds demonstrated dual antidiabetic activity, selected triterpenes proved to be potent antidiabetic agents in vitro.
- MeSH
- alpha-Glucosidases metabolism MeSH
- Cell Line MeSH
- Lamiaceae * chemistry MeSH
- Hypoglycemic Agents * pharmacology chemistry isolation & purification MeSH
- Glycoside Hydrolase Inhibitors pharmacology isolation & purification chemistry MeSH
- Mice MeSH
- Plant Components, Aerial chemistry MeSH
- Glucose Transporter Type 4 metabolism MeSH
- Plant Extracts chemistry pharmacology MeSH
- Triterpenes * pharmacology chemistry isolation & purification MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Single-photon optogenetics enables precise, cell-type-specific modulation of neuronal circuits, making it a crucial tool in neuroscience. Its miniaturization in the form of fully implantable wide-field stimulator arrays enables long-term interrogation of cortical circuits and bears promise for brain-machine interfaces for sensory and motor function restoration. However, achieving selective activation of functional cortical representations poses a challenge, as studies show that targeted optogenetic stimulation results in activity spread beyond one functional domain. While recurrent network mechanisms contribute to activity spread, here we demonstrate with detailed simulations of isolated pyramidal neurons from cats of unknown sex that already neuron morphology causes a complex spread of optogenetic activity at the scale of one cortical column. Since the shape of a neuron impacts its optogenetic response, we find that a single stimulator at the cortical surface recruits a complex spatial distribution of neurons that can be inhomogeneous and vary with stimulation intensity and neuronal morphology across layers. We explore strategies to enhance stimulation precision, finding that optimizing stimulator optics may offer more significant improvements than the preferentially somatic expression of the opsin through genetic targeting. Our results indicate that, with the right optical setup, single-photon optogenetics can precisely activate isolated neurons at the scale of functional cortical domains spanning several hundred micrometers.
- MeSH
- Cats MeSH
- Models, Neurological MeSH
- Cerebral Cortex physiology cytology MeSH
- Neurons physiology MeSH
- Optogenetics * methods MeSH
- Pyramidal Cells physiology MeSH
- Photic Stimulation methods MeSH
- Animals MeSH
- Check Tag
- Cats MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Ocrelizumab is a second generation recombinant humanized IgG1 monoclonal antibody used for the treatment of multiple sclerosis that selectively target B cells expressing the CD20 antigen. This study aimed to develop and validate a UPLC-MS/MS method for quantification of ocrelizumab in human serum, which can be used in clinical applications for therapeutic drug monitoring. The analysis of ocrelizumab was performed using a bottom-up approach on a liquid chromatography coupled to tandem mass spectrometry detection. The method involved immunoglobulin precipitation with cold methanol followed by peptide digestion with trypsin. The resulting specific peptides were separated on an Acquity UPLC BEH C18 column at 55 °C using gradient elution. The method was validated according to European Medicines Agency (EMEA) guidelines and demonstrated intra- and inter-assay precision with coefficients of variation ranging from 1.6 % to 6.1 % and accuracies between 90.2 % and 107.2 %. Stability testing, including autosampler, long-term and freeze-thaw stability, showed no more than 15 % variation. The method was successfully applied to 169 patient samples, revealing ocrelizumab concentrations ranging from 0.5 to 21.8 mg/L in patients on 6-month dosing regimen and 20.5-65.0 mg/L in 16 patients receiving an initial two-week dose of 300 mg. The newly developed UPLC-MS/MS method met all criteria for accuracy, precision and stability, confirming its suitability for clinical use in monitoring ocrelizumab levels in multiple sclerosis patients.
Autentizace rybích výrobků s využitím analýzy DNA vyžaduje zisk kvalitní DNA bez přítomnosti inhibitorů. V současné době jsou dostupné různé metody pro izolaci nukleových kyselin; pro svou rychlost a nenáročnost extrakčního postupu se staly velmi oblíbenými zejména silikátové centrifugační kolonky. Jejich nevýhodou však může být princip využívající záporný náboj DNA, který může být ovlivněn složením potravin, nebo jejich ucpání v důsledku špatné předúpravy vzorků. Cílem této práce bylo porovnat tři metody izolace DNA využívající různé principy (silikátové centrifugační kolonky, modifikované magnetické kuličky, cetyltrimethylamonium-bromid (CTAB) a chloroformová extrakce) a zhodnotit jejich vhodnost pro izolaci DNA z rybí svaloviny. Posuzovanými kritérii byla výtěžnost, čistota a amplifikovatelnost izolované DNA. Analyzována byla tkáň makrely obecné bez a s přídavkem přídatných látek běžně používaných při výrobě rybích produktů, konkrétně difosforečnanů (E 450) a barviv (E 110 a E 124), a následně byla vybraná metoda aplikována i na komerčně nabízené výrobky z ryb. Jako nejvhodnější se ukázala upravená metoda využívající detergent CTAB.
Authentication of fish products by DNA analysis requires the extraction of high quality DNA without the presence of inhibitors. Many nucleic acid isolation methods are currently available; silicate centrifugal columns have become very popular due to their speed and ease of extraction. However, their disadvantage may be the principle based on DNA charge, which may be affected by food composition, or clogging due to a poor sample pretreatment. The aim of this work was to compare three DNA isolation methods using different principles (silicate centrifugal columns, modified magnetic beads, Cetrimonium bromide and chloroform extraction) and to evaluate their suitability for DNA isolation from fish muscle. The criteria assessed were the recovery, purity and amplifiability of the isolated DNA. Mackerel tissue was analysed without and with the addition of additives commonly used in the manufacture of fish products, namely diphosphates (E 450) and colorants (E 110 and E 124), and the selected method was subsequently applied to commercial fish products. The modified method using the detergent CTAB proved to be the most suitable.
- MeSH
- Food Analysis methods MeSH
- DNA analysis isolation & purification MeSH
- Humans MeSH
- Polymerase Chain Reaction MeSH
- Seafood analysis MeSH
- Food Additives analysis MeSH
- Fish Products * analysis MeSH
- Fishes MeSH
- Nucleic Acid Amplification Techniques methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
The purpose of the present study was to purify and characterize the catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) enzyme from the local isolate of Pseudomonas putida. This enzyme catalyzes the initial reaction in the ortho-pathway for phenol degradation in various gram-negative bacteria, including the genus of Pseudomonas. Pseudomonads are commonly used in the biodegradation of xenobiotics due to their versatility in degrading a wide range of chemical compounds. Eighty-nine soil samples were taken from the contaminated soil of the Midland Refineries Company (MRC) of Al-Daura refinery area at Baghdad from April to August 2021. The samples were grown in a mineral salt medium containing 250 mg per L of phenol to test their ability to biodegrade phenol. The pH was adjusted to 8.0 at 30 °C using a shaking incubator for 24-48 h. A number of 62 (69.6%) isolates of the total number were able to degrade phenol efficiently. The findings of the VITEK system and the housekeeping gene 16S rDNA confirmed that out of the positive isolates for phenol degradation, 36 from 62 (58.06%) were identified as Pseudomonas spp. isolates. Those isolates were distributed as P. aeruginosa 30 (83.3%) and P. putida 6 (16.6%). The enzyme production capabilities of the isolates were evaluated, and the highest activity was 2.39 U per mg for the isolate No. 15 which it was identified as P. putida. The previous isolate was selected for enzyme production, purification, and characterization. The enzyme was purified using ion exchange and gel filtration chromatography, with a combined yield of 36.12% and purification fold of 15.42 folds. Using a gel filtration column, the enzyme's molar mass was calculated to be 69 kDa after purification. The purified enzyme was stable at 35 °C and a pH of 6.0.
- MeSH
- Bacterial Proteins metabolism genetics chemistry isolation & purification MeSH
- Biodegradation, Environmental * MeSH
- Phenol * metabolism MeSH
- Phylogeny MeSH
- Catechol 1,2-Dioxygenase * metabolism genetics MeSH
- Hydrogen-Ion Concentration MeSH
- Pseudomonas putida * enzymology genetics metabolism MeSH
- Soil Microbiology * MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
This is a retrospective cross-sectional study examining the association between unemployment, cancer type, treatment and total body fat percentage of childhood cancer survivors recruited at St. Anne's University Hospital in Brno, Czech Republic. A total of 55 survivors aged 18-49 who were in remission of cancer and fulfilled the criteria for body composition measurements by the BIA and completed questionnaires investigating their socioeconomic status, employment status, and history. There was a significant relationship between the employment status and central nervous system-directed treatment (c2(1) = 7.53, p = 0.006, Cramér's V = 0.38) and between the type of cancer and employment status (c2(3) = 7.83, p = 0.049, Cramér's V = 0.38), the highest unemployment rate was recorded for brain and spine survivors (72.7%) compared to survivors with other diagnosis (35.7%) (uLR(1) = 4.91, p = 0.027; OR = 4.80, 95% CI:1.10-20.86, p = 0.036); these survivors did not have a significantly different body fat percentage compared to survivors with other diagnoses (t(53) = 1.29, p = 0.202, Cohen's d = 0.41) Interestingly, the survivors reporting having a partner also had a significantly higher percentage of body fat (t(53) = 2.90, p = 0.005, Cohen's d = 0.81). A linear regression model was used to model the percentage of body fat in relation to a set of selected variables and the we observed a significant effect of sex (female vs male: b = 6.37, 95% CI: 1.82-10.93, p = 0.007), partnership status (yes vs no: b = 5.65, 95% CI: 0.67-10.62, p = 0.027) and category of diagnosis (Brain and spinal column tumors vs Other solid tumors: b = 12.40, 95% CI: 0.59-24.21, p = 0.040; Brain and spinal column tumors vs Lymphoma: b = 14.02, 95% CI: 2.06-25.97, p = 0.023). Employment status and risk of adiposity in childhood cancer survivors depends on the type of treatment and diagnosis group, which may significantly impact their lifestyle and overall quality of life after treatment. Trial registration: This study was registered on July 29, 2022, at ClinicalTrials.gov (NCT05481229).
- MeSH
- Adiposity MeSH
- Child MeSH
- Quality of Life MeSH
- Humans MeSH
- Central Nervous System Neoplasms * MeSH
- Neoplasms * epidemiology therapy MeSH
- Obesity MeSH
- Cancer Survivors * MeSH
- Cross-Sectional Studies MeSH
- Retrospective Studies MeSH
- Social Class MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
A case study on Sitagliptin drug products and Sitagliptin/Metformin drug products concerning contamination with N-nitrosamines was performed using two newly developed analytical methods for determination of N-nitroso-triazolopyrazine (NTTP; 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine) and its precursor triazolopyrazine (3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine). The method for determination of triazolopyrazine was previously unpublished, the method for determination of NTTP was published only for analysis of active pharmaceutical ingredient Sitagliptin and not the drug forms. Solving the N-nitrosamine contamination is requested by regulatory authorities all over the world and thus is vital for all pharmaceutical companies. The solution always requires a sensitive analytical method. Both newly developed methods use liquid chromatography coupled with mass spectrometry (single quadrupole analyzer in case of triazolopyrazine and triple quadrupole analyzer in case of NTTP). Separation of triazolopyrazine was achieved on a column Acquity CSH C18 using a mobile phase consisting of aqueous ammonium formate buffered at pH 4.2 and acetonitrile. Detection was performed using positive electrospray and selected ion monitoring at m/z 193. Separation of NTTP was achieved on a column Acquity HSS T3 using a mobile phase consisting of 0.1 % formic acid in water and methanol. Detection was performed using positive electrospray and multiple reaction monitoring at transitions m/z 222.15→42.05 (collision energy 17 eV) and m/z 222.15→192.15 (collision energy 11 eV). Two issues specific to NTTP and triazolopyrazine previously not described in scientific literature were successfully troubleshooted. Spontaneous degradation of Sitagliptin to triazolopyrazine and methyl (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoate was solved by using N,N-dimethylformamide as sample solvent during development of the method for quantitation of triazolopyrazine. A bad peak shape of NTTP due to the presence of rotamers of NTTP was successfully troubleshooted by increasing column temperature. Both methods were used during an optimization study of manufacturing of Sitagliptin and Sitagliptin/Metformin drug products. The goal of the study was to decrease NTTP content in the final drug product under the strict legislative limit set by Federal Drug Agency. The efficacy of several solutions was proven, but could not be fully disclosed due to Intellectual Property Protection policy of Zentiva. Instead, a brief review of recently published strategies to cope with N-nitrosamine contamination is presented.
The use of contaminated raw materials can lead to the transfer of mycotoxins into the final product, including beer. This study describes the use of the commercially available immunoaffinity column 11+Myco MS-PREP® and UPLC-MS/MS for the determination of mycotoxins in pale lager-type beers brewed in Czech Republic and other European countries. The additional aim of the work was to develop, optimize and validate this analytical method. Validation parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy were tested. The calibration curves were linear with correlation coefficients (R2 > 0.99) for all mycotoxins under investigation. The LOD ranged from 0.1 to 50 ng/L and LOQ from 0.4 to 167 ng/L. Recoveries of the selected analytes ranged from 72.2 to 101.1%, and the relative standard deviation under conditions repeatability (RSDr) did not exceed 16.3% for any mycotoxin. The validated procedure was successfully applied for the analysis of mycotoxins in a total of 89 beers from the retail network. The results were also processed using advanced chemometric techniques and compared with similar published studies. The toxicological impact was taken into account.
Úvod a cíl: Apikální periodontitida (AP) je zánětlivé onemocnění zubních periradikulárních tkání vyvolané bakteriální infekcí. Ke stanovení bakterií osídlujících kořenové kanálky jsou využívány různé metodické přístupy, analýza bakteriomu celého kořenového systému zubu postiženého AP je však zatím stále výzvou. Cílem naší přehledové práce bylo vytvořit literární rešerši zaměřenou na postupy pro odběr vzorku a metodiku pro analýzu bakteriomu a poté navrhnout vhodný metodický přístup k účelu studia etiopatogeneze tohoto onemocnění. Metodika: Po vyhledávání v databázi PubMed jsme do rešerše vybrali pouze publikační výstupy typu původní práce, ve kterých byla analyzována bakteriální DNA lidských zubů. Výsledky: Metodicky se studie mezi sebou velmi liší, a to jak způsobem odběru vzorku, izolací DNA, tak i samotnou analýzou bakteriální DNA. Častým způsobem odběru vzorku je využití sterilních endodontických papírových čepů. Tento způsob odběru vzorku je sice vhodný v rámci klinické praxe, avšak pro komplexní analýzu prostředí kořenového systému je považován za nedostatečný, a to kvůli samotné morfologii zubu a přítomnosti ramifikací. Jiným způsobem odběru vzorku je resekce kořenového hrotu pomocí sterilních fréz a následné rozemletí apexu nebo provedení stěru sterilními endodontickými papírovými čepy. Ke stanovení bakteriomu je tak využívána pouze apikální část zubu, tudíž bakterie, které kolonizují koronární část zubu a podílejí se na etiopatogenezi onemocnění, nemohou být analyzovány. V recentních studiích je využívána metoda, při které je celý extrahovaný zub postižený AP nadrcen na jemný homogenní prach pomocí kryogenního mletí. Z prachu nadrceného zubu je možné stanovit komplexní bakteriom kořenového systému i dřeňové dutiny, a proto se tento způsob z pohledu přípravy vzorku pro experimentální studii jeví jako optimální. Nejčastěji je při izolaci DNA využívána efektivní kolonková metoda různými purifikačními soupravami a k následné analýze DNA slouží většinou metodiky založené na principu polymerázové řetězové reakce. Sekvenování variabilních oblastí genu pro 16S rRNA je v dnešní době již zlatým standardem pro kategorizaci bakterií a charakterizaci bakteriálních komunit. Závěr: Ke studiu bakteriomu AP se jeví jako nejvhodnější použít vzorky extrahovaných zubů a bezprostředně je zamrazit bez dalších preanalytických kroků. Nadrcený zub je při dodržení sterilních podmínek při kryogenním mletí vhodnou matricí pro izolaci mikrobiální DNA komerčně dostupnými kity. V současné době je pro stanovení bakteriomu a získání informace o relativní abundanci bakteriálních rodů, a to z analytického i ekonomického hlediska, sekvenování nové generace nejlepší volbou.
Introduction, aim: Apical periodontitis (AP) is an inflammatory disease of the dental periradicular tissues caused by a bacterial infection. Various methodological approaches are used to determine the bacteria inhabiting the root canals, however, the analysis of the entire root system of a tooth affected by AP still remains a challenge. The aim of our study was to perform a literature search focused on sample collection procedures and methodologies for bacteriome analysis, and then propose a suitable methodological approach for the purpose of studying the etiopathogenesis of this disease. Methods: After searching the PubMed database, we selected only publications of the original work type in which the bacterial DNA of human teeth was analyzed, for the search. Results: Methodologically, the studies differ greatly, in terms of sample collection, DNA isolation, and bacterial DNA analysis itself. A common method of sample collection is the use of sterile endodontic paper points. Although this method of sampling is suitable in clinical practice, it is considered insufficient for a comprehensive analysis of the environment of the root canal system, due to the morphology of the tooth itself and the presence of ramifications. Another method of sampling is resection of the root tip using sterile burs and subsequent grinding of the apex or smearing with sterile endodontic paper pins. Only the apical part of the tooth is used to determine the bacteriome, therefore bacteria that colonize the coronal part of the tooth and participate in the etiopathogenesis of the disease cannot be analyzed. In recent studies, a method is used in which the entire extracted tooth affected by AP is ground into a fine homogeneous powder using cryogenic grinding. It is possible to determine the complex bacteriome of the root canal system and the pulp chamber from the dust of a crushed tooth, and therefore this method seems optimal for the sample preparation from an experimental study point of view. Most often, an effective column method with various purification kits is used for DNA isolation, and for subsequent DNA analysis, methodologies based on the principle of the polymerase chain reaction are mostly used. Sequencing the variable regions of the gene for 16S rRNA is nowadays already the gold standard for categorizing bacteria and characterizing bacterial communities. Conclusion: To study the AP bacteriome, it seems most appropriate to use extracted tooth samples and immediate freezing of the sample without further pre-analytical steps. The crushed tooth is a suitable matrix for the isolation of microbial DNA with commercially available kits, provided that sterile conditions are maintained during cryogenic grinding. Currently, next-generation sequencing is the best choice for determining the bacteriome and obtaining information about the relative abundance of bacterial genera, both analytically and economically.
- Keywords
- kryomlýnek,
- MeSH
- DNA isolation & purification MeSH
- Dental Pulp Cavity microbiology pathology MeSH
- Humans MeSH
- Periapical Periodontitis * microbiology pathology therapy MeSH
- Sequence Analysis, DNA MeSH
- Root Canal Therapy MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
The continuous expansion of research in the field of stable carboranes and their wide potential in the drug design require carrying out fundamental studies regarding their chiral separations. Although supercritical fluid chromatography (SFC) is a viable technique for fast enantioseparations, no investigation concerning boron cluster compounds has been done yet. We aimed at the development of a straightforward method enabling chiral separations of racemic mixtures of anionic cluster carboranes and metallacarboranes that represent an analytical challenge. The fast gradient screening testing nine polysaccharide-based columns was used. The key parameters affecting the selectivity were the type of chiral selector, the type of alcohol, and the base in cosolvent. Moreover, the addition of acetonitrile or water to the cosolvent was identified as an effective tool for decreasing the analysis time while preserving the resolution. After the optimization, the chiral separations of 19 out of 20 selected compounds were achieved in less than 10 min. These results demonstrate the clear advantage of SFC over chiral separations using HPLC in terms of both analysis time and structural variety of successfully separated compounds.
