Sample purification
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The effect of the antibiotic tetracycline on the growth and photosynthetic activity of the diatoms Cyclotella caspia Grunow, 1878, and Thalassiosira weissflogii (Grunow) G.A. Fryxell & Hasle, 1977, has been studied. The rate of tetracycline concentration decreases in the medium with and without has been estimated. The expediency and prospects of using diatoms as promising objects for water purification from tetracycline are shown. It was found that the rate and efficiency of tetracycline removal from the medium in the presence of algae depends on the initial content of the pollutant in the medium. The maximum efficiency of antibiotic removal is observed at concentrations of the pollutant provoking the hormesis growth of algae at 5 mg/L in C. caspia and 10 mg/L in T. weissflogii. In samples with C. caspia and tetracycline, the residual antibiotic content in the medium was 10-14% lower than in samples without algae. In the experiment with T. weissflogii, this indicator reached 15-16%. At concentrations of the pollutant, 2 and 10 mg/L in C. caspia and 2 and 5 mg/L in T. weissflogii, the effectiveness of removing the antibiotic by algae was lower. The residual content of tetracycline in the medium was on average 8-11% lower than in samples without algae. At antibiotic concentrations of 15 and 20 mg/L, the rate of its removal in samples with and without algae did not significantly differ. This result is obviously due to the toxic effect of tetracycline on cells, leading to pronounced inhibition of algae growth and/or death.
Applications like drug development need simple and streamlined methods to process samples from 96-well cell culture plates for gene expression measurements. Unfortunately, current options are expensive for such processing. Therefore, our aim was to develop a method that would allow streamlined analysis of mRNA from 96-well cell culture plates while being relatively cheap and simple. We developed a method based on the qPCR 'Cells-to-cDNA' approach and validated it against commercially available kits using the same approach or spin columns-based RNA purification. For this purpose, we conducted a series of comparisons of gene expression from peripheral blood mononuclear cells, SK-HEP-1 and U-87 cell cultures in 96-well plates. Our final method involved lysing cells with 25-100 μl solution of 0.5% SDS, 10 mM DTT, 1 mg ml-1 proteinase K dissolved in water, 1 h incubation at 50°C, followed by proteinase K inactivation at 90°C for 5 min and lysate neutralization with 1 : 1 dilution by 20% Tween 20 solution. Reverse transcription and qPCR were carried out using standard methods. This method showed a mean reduction of Ct ± s.d. value by 2.4 ± 1.3 compared with the 'Cells-to-cDNA' kit and by 1.4 ± 0.5 compared with the RNA purification kit with lower variability.
- MeSH
- analýza nákladů a výnosů MeSH
- buněčné kultury metody ekonomika MeSH
- komplementární DNA * genetika MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- leukocyty mononukleární cytologie metabolismus MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- stanovení celkové genové exprese metody ekonomika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Proteomics provides an understanding of biological systems by enabling the detailed study of protein expression profiles, which is crucial for early disease diagnosis. Microfluidic-based proteomics enhances this field by integrating complex proteome analysis into compact and efficient systems. This review focuses on developing microfluidic chip structures for proteomics, covering on-chip sample pretreatment, protein extraction, purification, and identification in recent years. Furthermore, our work aims to inspire researchers to select proper methodologies in designing novel, efficient assays for proteomics applications by analyzing trends and innovations in this field.
- MeSH
- biosenzitivní techniky přístrojové vybavení metody MeSH
- design vybavení MeSH
- laboratoř na čipu * MeSH
- lidé MeSH
- mikrofluidika metody MeSH
- mikrofluidní analytické techniky přístrojové vybavení MeSH
- proteiny analýza izolace a purifikace MeSH
- proteom analýza izolace a purifikace chemie MeSH
- proteomika * metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The level of the human body's burden of benzophenone and camphor ultraviolet (UV) filters can be estimated from their urinary levels. The present study describes the implementations and validation of the sensitive analytical method for the analysis of seven benzophenone and two camphor UV filters in urine. Sample preparation includes overnight enzymatic hydrolysis and ethyl acetate extraction followed by purification by dispersive solid-phase extraction using a sorbent Z-Sep. For the analysis, ultra-high performance liquid chromatography coupled with tandem mass spectrometry was used. Validation was performed using a Standard Reference Material® 3673 and an artificially contaminated urine sample. Target analyte recoveries ranged from 79-113% with repeatability expressed as a relative standard deviation of 2-15%. The limits of quantification were between 0.001 and 0.100 ng/mL in urine. This method was subsequently applied to examine the urine samples collected from Czech women. The analytes benzophenone-1 and 4-hydroxy-benzophenone were the most common analytes present in 100% of the samples, whereas benzophenone-3 was quantified in only 90% of the urine samples. The other four determined benzophenone derivatives were quantified in ≤33% of the samples. The derivatives of camphor were not detected in any samples. This method could be applied in biomonitoring studies.
- Publikační typ
- časopisecké články 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
- bakteriální proteiny metabolismus genetika chemie izolace a purifikace MeSH
- biodegradace * MeSH
- fenol * metabolismus MeSH
- fylogeneze MeSH
- katechol-1,2-dioxygenasa * metabolismus genetika MeSH
- koncentrace vodíkových iontů MeSH
- Pseudomonas putida * enzymologie genetika metabolismus MeSH
- půdní mikrobiologie * MeSH
- RNA ribozomální 16S genetika MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
Laccase-producing fungus (MY3) was successfully isolated from soil samples collected from Mansoura Governorate, Egypt. This fungal isolate has shown a high laccase production level over other isolated fungi. The identity of this isolate was determined by the molecular technique 18SrRNA as Curvularia lunata MY3. The enzyme purification was performed using ammonium sulfate precipitation followed by Sephacryl S-200 and DEAE-Sepharose column chromatography. The denatured enzyme using SDS-PAGE had a molar mass of 65 kDa. The purified laccase had an optimum temperature at 40 °C for enzyme activity with 57.3 kJ/mol activation energy for 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidation. The enzyme had an optimum pH of 5.0, and it has shown a high stability at the acidic range (4.5 to 5.5). Mn2+ and Mg2+ ions enhanced the enzyme activity, while most of the enzyme activity was inhibited by Hg2+. Some compounds such as 2-mercaptoethanol, L-cysteine, and sodium azide at a concentration of 10 mmol/L had shown a high suppression effect on the enzyme activity. The enzyme strongly oxidized ABTS and syringaldazine and moderately oxidized DMP and guaiacol. The antimicrobial activity of the purified enzyme towards three pathogenic strains (Escherichia coli ATCC-25922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC-10231) was evaluated for the potential use as an antimicrobial therapeutic enzyme.
Colorectal cancer (CRC) is the second most prevalent cancer type worldwide, which highlights the urgent need for non-invasive biomarkers for its early detection and improved prognosis. We aimed to investigate the patterns of long non-coding RNAs (lncRNAs) in small extracellular vesicles (sEVs) collected from low-volume blood serum specimens of CRC patients, focusing on their potential as diagnostic biomarkers. Our research comprised two phases: an initial exploratory phase involving RNA sequencing of sEVs from 76 CRC patients and 29 healthy controls, and a subsequent validation phase with a larger cohort of 159 CRC patients and 138 healthy controls. Techniques such as dynamic light scattering, transmission electron microscopy, and Western blotting were utilized for sEV characterization. Optimized protocol for sEV purification, RNA isolation and preamplification was applied to successfully sequence the RNA content of sEVs and validate the results by RT-qPCR. We successfully isolated sEVs from blood serum and prepared sequencing libraries from a low amount of RNA. High-throughput sequencing identified differential levels of 460 transcripts between CRC patients and healthy controls, including mRNAs, lncRNAs, and pseudogenes, with approximately 20% being lncRNAs, highlighting several tumor-specific lncRNAs that have not been associated with CRC development and progression. The validation phase confirmed the upregulation of three lncRNAs (NALT1, AL096828, and LINC01637) in blood serum of CRC patients. This study not only identified lncRNA profiles in a population of sEVs from low-volume blood serum specimens of CRC patients but also highlights the value of innovative techniques in biomolecular research, particularly for the detection and analysis of low-abundance biomolecules in clinical samples. The identification of specific lncRNAs associated with CRC provides a foundation for future research into their functional roles in cancer development and potential clinical applications.
Osteoporosis is a multifactorial disease influenced by genetic and environmental factors, which contributes to an increased risk of bone fracture, but early diagnosis of this disease cannot be achieved using current techniques. We describe a generic platform for the targeted electrochemical genotyping of SNPs identified by genome-wide association studies to be associated with a genetic predisposition to osteoporosis. The platform exploits isothermal solid-phase primer elongation with ferrocene-labeled nucleoside triphosphates. Thiolated reverse primers designed for each SNP were immobilized on individual gold electrodes of an array. These primers are designed to hybridize to the SNP site at their 3'OH terminal, and primer elongation occurs only where there is 100% complementarity, facilitating the identification and heterozygosity of each SNP under interrogation. The platform was applied to real blood samples, which were thermally lysed and directly used without the need for DNA extraction or purification. The results were validated using Taqman SNP genotyping assays and Sanger sequencing. The assay is complete in just 15 min with a total cost of 0.3€ per electrode. The platform is completely generic and has immense potential for deployment at the point of need in an automated device for targeted SNP genotyping with the only required end-user intervention being sample addition.
- Publikační typ
- časopisecké články MeSH
Fungi are producers of lignolytic extracellular enzymes which are used in industries like textile, detergents, biorefineries, and paper pulping. This study assessed for the production, purification, and characterization of novel p-diphenol oxidase (PDO; laccase) enzyme from lignolytic white-rot fungal isolate. Fungi samples collected from different areas of Pakistan were initially screened using guaiacol plate method. The maximum PDO producing fungal isolate was identified on the basis of ITS (internal transcribed spacer sequence of DNA of ribosomal RNA) sequencing. To get optimum enzyme yield, various growth and fermentation conditions were optimized. Later PDO was purified using ammonium sulfate precipitation, size exclusion, and anion exchange chromatography and characterized. It was observed that the maximum PDO producing fungal isolate was Schizophyllum commune (MF-O5). Characterization results showed that the purified PDO was a monomeric protein with a molecular mass of 68 kDa and showed stability at lower temperature (30 °C) for 1 h. The Km and Vmax values of the purified PDO recorded were 2.48 mM and 6.20 U/min. Thermal stability results showed that at 30 °C PDO had 119.17 kJ/K/mol Ea value and 33.64 min half-life. The PDO activity was stimulated by Cu2+ ion at 1.0 mM showing enhanced activity up to 111.04%. Strong inhibition effect was noted for Fe2+ ions at 1 mM showing 12.04% activity. The enzyme showed stability against 10 mM concentration oxidizing reducing agents like DMSO, EDTA, H2O2, NaOCl, and urea and retained more than 75% of relative activity. The characterization of purified PDO enzyme confirmed its tolerance against salt, metal ions, organic solvents, and surfactants indicating its ability to be used in the versatile commercial applications.
miRNA expression in triple-negative breast cancers (TNBC) has mainly been studied from a methodological viewpoint. However, it has not been considered that miRNA expression profile may be associated with a specific morphological entity inside every tumor. The verification of this hypothesis on a set of 25 TNBCs was the subject of our previous work, where we confirmed specific expression of the studied miRNAs in 82 samples of different morphologies including inflammatory infiltrate, spindle cell, clear cell, and metastases after RNA extraction and purification as well as microchip and biostatistical analysis. In the current work, we demonstrate a low suitability of in situ hybridization method for miRNA detection compared to RT-qPCR, and in detail discuss the biological role of 8 miRNAs with the most significant changes of expression.
- MeSH
- lidé MeSH
- mikro RNA * genetika metabolismus MeSH
- triple-negativní karcinom prsu * genetika patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
