Search Results (22)

Background: This study presents a sensitive method for the simultaneous determination of organic acids, flavonoids, and amino acids in Mume Fructus (MF) using ultra-performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry (UPLC-QTRAP-MS/MS). Methods: Analysis was performed on a UPLC system (Shimadzu, Kyoto, Japan) equipped with a quaternary pump solvent management system, an online degasser, a triple-quadrupole mass detector, and an autosampler. An Agilent ZORBAX SB-C18 column (3.0 mm × 100 mm, 1.8 µm) was used for chromatographic analyses. The mobile phase was distributed between 0.2% aqueous formic acid (A) and 0.2% formic acid acetonitrile (B) at a velocity of 0.2 mL/min. The gradient evolution protocol was 0–2 min at 90–70% B; 3–7 min at 70–50% B; 7–10 min at 50–20% B; 10–14.5 min at 20–90% B; and 14.5–17 min at 10% B. Results: The method was validated for matrix effects, linearity, limits of detection/quantification, precision, repeatability, stability, and recovery of target components. It effectively determined all target compounds in 12 MF batches from different drying methods. Conclusions: Principal component analysis (PCA) of 47 active components was conducted to evaluate MF quality comprehensively. The proposed method serves as a reliable approach for assessing the consistency of MF’s quality and therapeutic efficacy. Full article

Dimorphism of sex chromosomes often leads to a need for dosage compensation. In eutherian mammals, XIST, a long non-coding RNA, is expressed from the X chromosome that will be silenced, triggering X-chromosome inactivation (XCI). XIST originated from the ancestral protein-coding Lnx3 gene with contributions from various mobile elements that contributed to the striking domains of tandem repeats within the first and sixth exons. Modular domains of XIST are now involved in recruiting heterochromatic marks and proteins essential for XCI initiation and maintenance. This review presents a comparative analysis of human XIST with five other eutherian mammals—chimpanzees, cats, pigs, sheep, and mice—examining conservation across exons as well as the tandem repeats. Notably, repeats exhibited higher conservation than exons, underscoring their functional importance. Additionally, a species-specific G repeat, previously described in pigs, was also identified in sheep and cats. These findings provide insights into the domains of XIST, a cis-acting silencer that has been used to proposed to alleviate the impact of a supernumerary chromosome in Down syndrome. Full article

Oxysterols, as metabolites of cholesterol, play a key role in cholesterol homeostasis, autophagosome formation, and regulation of immune responses. Disorders in oxysterol metabolism are closely related to the pathogenesis of neurodegenerative diseases. To systematically investigate the profound molecular regulatory mechanisms of neurodegenerative diseases, it is necessary to quantify oxysterols and their metabolites in central and peripheral biospecimens simultaneously and accurately. However, there are a lot of unsolved problems with the existing methods, such as the hindrance of applying a single method to different biological specimens or the challenge of simultaneous quantification due to differential groups on the ends of the oxysterol side chains. Herein, according to the physicochemical properties and structure of oxysterols, an optimized liquid chromatography-tandem mass spectrometry method for the quantification of oxysterols was established by optimizing the sample preparation process, chromatographic conditions, mobile phase pH, and solvent selection. Seven oxysterols were detected by this method, including 27-hydroxycholesterol, 7α-hydroxycholesterol, 7α,27-dihydroxycholesterol, 7-dehydrocholesterol, 7α-hydroxy-3-oxo-4-cholestenoic acid, 3-hydroxy-5-cholestenoic acid, and 24(S)-hydroxycholesterol. Non-derivatization extraction with methyl tert-butyl ether was used for different biospecimens, followed by simultaneous chromatographic separation of oxysterols on a phenyl hexyl column. By repeated validation, this method exhibited satisfactory linearity, precision, recovery, sensitivity, repeatability, and stability, and it was successfully applied to the detection of oxysterols in the plasma, cerebral cortex, and liver of mouse. In summary, our optimized method enables concurrent analysis and quantification of oxysterols and their metabolites in various biospecimens, presenting a broad range of applicability. Full article

The bacterium Deinococcus radiodurans is known to efficiently and accurately reassemble its genome after hundreds of DNA double-strand breaks (DSBs). Only at very large amounts of radiation-induced DSBs is this accuracy affected in the wild-type D. radiodurans, causing rearrangements in its genome structure. However, changes in its genome structure may also be possible during the propagation and storage of cell cultures. We investigate this possibility by listing structural differences between three completely sequenced genomes of D. radiodurans strains with a recent common ancestor—the type strain stored and sequenced in two different laboratories (of the ATCC 13939 lineage) and the first sequenced strain historically used as the reference (ATCC BAA-816). We detected a number of structural differences and found the most likely mechanisms behind them: (i) transposition/copy number change in mobile interspersed repeats—insertion sequences and small non-coding repeats, (ii) variable number of monomers within tandem repeats, (iii) deletions between long direct DNA repeats, and (iv) deletions between short (4–10 bp) direct DNA repeats. The most surprising finding was the deletions between short repeats because it indicates the utilization of a less accurate DSB repair mechanism in conditions in which a more accurate one should be both available and preferred. The detected structural differences, as well as SNPs and short indels, while being important footprints of deinococcal DNA metabolism and repair, are also a valuable resource for researchers using these D. radiodurans strains. Full article

Aerial–aquatic vehicles (AAVs) hold great promise for marine applications, offering adaptability to diverse environments by seamlessly transitioning between underwater and aerial operations. Nevertheless, the design of AAVs poses inherent challenges, owing to the distinct characteristics of different fluid media. This article introduces a novel solution in the form of a tandem dual-rotor aerial–aquatic vehicle, strategically engineered to overcome these challenges. The proposed vehicle boasts a slender and streamlined body, enhancing its underwater mobility while utilizing a tandem rotor for aerial maneuvers. Outdoor scene tests were conducted to assess the tandem dual-rotor AAV’s diverse capabilities, including flying, hovering, and executing repeated cross-media locomotion. Notably, its versatility was further demonstrated through swift surface swimming on water. In addition to aerial evaluations, an underwater experiment was undertaken to evaluate the AAV’s ability to traverse narrow underwater passages. This capability was successfully validated through the creation of a narrow underwater gap. The comprehensive exploration of the tandem dual-rotor AAV’s potential is presented in this article, encompassing its foundational principles, overall design, simulation analysis, and avionics system design. The preliminary research and design outlined herein offer a proof of concept for the tandem dual-rotor AAV, establishing a robust foundation for AAVs seeking optimal performance in both water and air environments. This contribution serves as a valuable reference solution for the advancement of AAV technology. Full article

The amount of macrolide (MAL) residues in aquatic products, including oleandomycin (OLD), erythromycin (ERM), clarithromycin (CLA), azithromycin (AZI), kitasamycin (KIT), josamycin (JOS), spiramycin (SPI), tilmicosin (TIL), tylosin (TYL), and roxithromycin (ROX), was determined using solid-phase extraction and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The residues were extracted with 1% ammonia acetonitrile solution and purified by neutral alumina adsorption. Chromatographic separation was completed on an ACQUITY UPLC BEH C₁₈ column with acetonitrile–0.1% formic acid aqueous solution as the mobile phase, and mass spectrometry detection was performed by multiple reaction monitoring scanning with the positive mode in an electrospray ion source (ESI⁺). Five isotopically labeled compounds were used as internal standards for quality control purposes. The findings indicated that across the mass concentration span of 1.0–100 μg/L, there was a strong linear correlation (R² > 0.99) between the concentration and instrumental response for the 10 MALs. The limit of detection of UPLC-MS/MS was 0.25–0.50 μg/kg, and the limit of quantitation was 0.5–1.0 μg/kg. The added recovery of blank matrix samples at standard gradient levels (1.0, 5.0, and 50.0 μg/kg) was 83.1–116.6%, and the intra-day precision and inter-day precisions were 3.7 and 13.8%, respectively. The method is simple and fast, with high accuracy and good repeatability, in line with the requirements for accurate qualitative and quantitative analysis of the residues for 10 MALs in aquatic products. Full article

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections worldwide. Biofilm production, antibiotic resistance, and a wide range of virulence factors contribute to their persistence in nosocomial environments. We describe an outbreak caused by a multidrug-resistant P. aeruginosa strain in an ICU. Antibiotic susceptibility was determined and bla_PER-1 and qnrVC were amplified via PCR. Clonality was determined using PFGE and biofilm formation was studied with a static model. A combination of antibiotics was assessed on both planktonic cells and biofilms. WGS was performed on five isolates. All isolates were clonally related, resistant to ceftazidime, cefepime, amikacin, and ceftolozane-tazobactam, and harbored bla_PER-1; 11/19 possessed qnrVC. Meropenem and ciprofloxacin reduced the biofilm biomass; however, the response to antibiotic combinations with rifampicin was different between planktonic cells and biofilms. WGS revealed that the isolates belonged to ST309 and serotype O11. bla_PER-1 and qnrVC6 were associated with a tandem of ISCR1 as part of a complex class one integron, with aac(6′)-Il and ltrA as gene cassettes. The structure was associated upstream and downstream with Tn4662 and flanked by direct repeats, suggesting its horizontal mobilization capability as a composite transposon. ST309 is considered an emerging high-risk clone that should be monitored in the Americas. Full article

Infigratinib, a protein kinase inhibitor employed in the therapeutic management of cholangiocarcinoma, was subjected to various stress conditions, including hydrolytic (acidic and alkaline), oxidative, photolytic, and thermal stress, in accordance with the rules established by the International Council for Harmonization. A cumulative count of five degradation products was observed. The application of the Quality by Design principle was utilized in the development of a rapid and specific separation method for Infigratinib and its degradation products. The methodology employed in this study was derived from an experimental design approach, which was utilized to examine the critical process parameters associated with chromatographic systems. The reversed-phase high-performance liquid chromatography technique, employing a C18 column and a mobile phase composed of a gradient mixture of 25 mM ammonium acetate buffer at pH 6.0 and acetonitrile, successfully facilitated the chromatographic separation. The methodology was expanded to include the utilization of UPLC-quadrupole tandem mass spectrometry in order to conduct a comprehensive analysis of the structural properties and characterize the degradation products. Overall, five degradation products were found in different stress conditions. The method was verified at certain working points, wherein a linearity range (5.0–200.0 µg/mL) was developed and other parameters such as accuracy, repeatability, selectivity, and system suitability were evaluated. Finally, the toxicity and mutagenicity of Infigratinib and its degradation products were predicted using in silico software, namely DEREK Nexus^® (version 6.2.1) and SARAH Nexus^® (version 3.2.1). Various toxicity endpoints, including chromosomal damage, were predicted. Additionally, two degradation products were also predicted to be mutagenic. Full article

Polylactic acids (PLAs) are synthetic polymers composed of repeating lactic acid subunits. For their good biocompatibility, PLAs have been approved and widely applied as pharmaceutical excipients and scaffold materials. Liquid chromatography-tandem mass spectrometry is a powerful analytical tool not only for pharmaceutical ingredients but also for pharmaceutical excipients. However, the characterization of PLAs presents particular problems for mass spectrometry techniques. In addition to their high molecular weights and wide polydispersity, multiple charging and various adductions are intrinsic features of electrospray ionization. In the present study, a strategy combining of differential mobility spectrometry (DMS), multiple ion monitoring (MIM) and in-source collision-induced dissociation (in source-CID) has been developed and applied to the characterization and quantitation of PLAs in rat plasma. First, PLAs will be fragmented into characteristic fragment ions under high declustering potential in the ionization source. The specific fragment ions are then screened twice by quadrupoles to ensure a high signal intensity and low interference for mass spectrometry detection. Subsequently, DMS technique has been applied to further reduce the background noise. The appropriately chosen surrogate specific precursor ions could be utilized for the qualitative and quantitative analysis of PLAs, which provided results with the advantages of low endogenous interference, sufficient sensitivity and selectivity for bioassay. The linearity of the method was evaluated over the concentration range 3–100 μg/mL (r² = 0.996) for PLA 20,000. The LC-DMS-MIM coupled with in source-CID strategy may contribute to the pharmaceutical studies of PLAs and the possible prospects of other pharmaceutical excipients. Full article

Steroid hormones, such as 17β-testosterone, 11-ketotestorenone and 17β-estradiol, play an essential role not only in reproductive function but also are potential biomarkers of numerous additional functions in teleost fish. The presence of endocrine disruptor compounds in aquatic ecosystems has raised concern about their effect on hormone levels in fish target organs. Since hormones are present in very low concentrations in biological material, their determination still remains a challenge. A new analytical procedure has been developed to determine 17β-testosterone, 11-ketotestosterone and 17β-estradiol in the sea trout female and male gonads by liquid chromatography-tandem mass spectrometry (LC-MS/MS) system equipped with an ESI source operating in both positive and negative mode. Chromatographic separation of analytes was accomplished in Poroshell 120 EC-C18 (150 mm × 2.1 mm, 2.7 µm) column under isocratic elution conditions. The mobile phase consisted of acetonitrile, methanol and water (20:50:30/v/v/v) at a flow rate of 0.2 mL/min. Analytes were extracted from the gonad matrix with ethyl acetate, and co-extractives impurities were successfully removed by QuEChERS (quick, easy, cheap, effective, rugged and safe) method. The procedure was validated with good sensitivity, linearity, accuracy, and precision. Limits of quantifications were from 0.15 to 0.75 ng/g, linearity was obtained with correlation coefficient R > 0.99, accuracy was from 94.0 to 109.5%, precision expressed as RSD ranged from 1.7 to 27.2% (repeatability) and from 2.2 to 37.1% (reproducibility). Finally, the method was applied to determining 17β-testosterone, 11-ketotestosterone and 17β-estradiol in real samples of the female and male sea trout gonads, 8 and 22 samples, respectively. Full article

Monocytes are circulating blood cells that rapidly mobilize to inflamed sites where they serve diverse effector functions shaped in part by microenvironmental cues. The establishment of specific glycosylation patterns on the immune cell glycocalyx is fundamental to direct the inflammatory response, but relatively little is known about the mechanisms whereby the microenvironment controls this process. Here, we report that galectins differentially participate in remodeling the surface glycosylation of human primary CD14⁺CD16⁻ monocytes under proinflammatory conditions. Using a lectin array on biotinylated protein, we found that the prototypic galectin-1 negatively influenced the expression of galactose epitopes on the surface of monocytic cells. On the other hand, the tandem-repeat galectin-8 and, to a certain extent, the chimeric galectin-3 promoted the expression of these residues. Jacalin flow cytometry and pull-down experiments further demonstrated that galectin-8 causes a profound upregulation of mucin-type O-glycosylation in cell surface proteins from primary monocytes and THP-1 cells. Overall, these results highlight the emerging role of the galectin signature on inflamed tissues and provide new insights into the contribution of extracellular galectins to the composition of the glycocalyx in human monocytes. Full article

Cyanobacteria are a diverse group of oxygenic photosynthetic prokaryotes, which are believed to be one of the oldest life forms on Earth. They live in a wide range of ecosystems and withstand extreme environmental conditions. An important proportion of cyanobacteria is known to be producers of harmful cyanotoxins, which are toxic secondary metabolites that can impact the ecosystem and human health. The oral route is one of the main ways whereby humans can be exposed to cyanotoxins. Therefore, the consumption of contaminated algae-based food supplements is becoming more relevant due to its upsurge, which underlines the importance of controlling these toxins in this kind of products. This work describes the simultaneous determination of seven cyanotoxins belonging to three different classes: the cyclic peptides microcystin-LR (MC-LR), microcystin-RR (MC-RR) and nodularin (NOD); the alkaloid anatoxin-a (ANA) and three non-protein amino acids isomers β-methylamine-L-alanine (BMAA), 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl)glycine (AEG). These have been determined in spirulina-derived food supplements using a novel solid–liquid extraction coupled with a solid phase extraction procedure for clean up and preconcentration (SLE-tandem-SPE) and analysis by hydrophilic interaction liquid chromatography with tandem mass spectrometry detection (HILIC-MS/MS). A SeQuant® Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) column (EMD Millipore, Billerica, MA, USA) was employed to achieve the chromatographic separation in less than 12 min using water and acetonitrile, both acidified with 0.3% of formic acid, as mobile phase. Previously, an SLE was developed, using 4 mL of aqueous 5% formic acid to extract the most polar compounds, followed by 4 mL of 80% MeOH. Both extracts were combined and submitted to a tandem-SPE using mixed-mode cation exchange (MCX) and Strata-X cartridges. Elution from both cartridges was performed using 10% NH₃·H₂O in MeOH. Method validation was carried out in terms of linearity, limit of detection (LOD) and quantification (LOQ), recoveries, matrix effect and repeatability and intermediate precision. LOQs in the range of 50–300 µg·kg⁻¹ and recoveries ranging between 64.2% and 102.9% with an associated relative standard deviation < 19.2% were achieved. Satisfactory precision was obtained with RSD values lower than 19.6% in all cases, with the exception of BMAA, which reported the highest RSD values, reaching 25.1%. The method was satisfactorily applied to determine the occurrence of cyanotoxins in blue green algae (BGA) dietary supplements. DAB was the most frequently detected cyanotoxin, at concentrations up to 2408 µg·kg⁻¹, and AEG was found in few samples at concentrations up to 194 µg·kg⁻¹. However, MC-LR and MC-RR were found in one sample at concentration levels higher than 5 mg·kg⁻¹, which underlines the need to control these substances in these matrices. Full article

The extensive use of herbicides, such as glyphosate and glufosinate, in crop production during recent decades has raised concerns about human exposure. Nevertheless, analysis of trace levels of these herbicides in human biospecimens has been challenging. Here, we describe a method for the determination of urinary glyphosate, its degradation product aminomethylphosphonic acid (AMPA), and glufosinate using liquid chromatography-tandem mass spectrometry (LC–MS/MS). The method was optimized using isotopically labelled internal standards (¹³C₂, ¹⁵N-glyphosate, ¹³C, ¹⁵N, D₂-AMPA, and D₃-glufosinate) and solid-phase extraction (SPE) with cation-exchange and anion-exchange cartridges. The method provides excellent chromatographic retention, resolution and peak shape of target analytes without the need for strong acidic mobile phases and derivatization steps. The instrument linearity was in the range of 0.1–100 ng/mL, with R > 0.99 in the matrix for all analytes. The method detection limits (MDLs) and the method quantification limits (MQLs) were in the ranges of 0.12 (AMPA and glufosinate)–0.14 (glyphosate) ng/mL and 0.40 (AMPA)–0.48 (glyphosate) ng/mL, respectively. The recoveries of analytes spiked into urine matrix ranged from 79.1% to 119%, with coefficients of variation (CVs) of 4–10%. Repeated analysis of samples for over 2 weeks showed intra-day and inter-day analytical variations of 3.13–10.8% and 5.93–12.9%, respectively. The matrix effects for glyphosate, AMPA, and glufosinate spiked into urine matrix averaged −14.4%, 13.2%, and 22.2%, respectively. The method was further validated through the analysis of external quality assurance proficiency test (PT) urine samples. The method offers optimal sensitivity, accuracy, and precision for the urine-based assessment of human exposure to glyphosate, AMPA, and glufosinate. Full article

The increasing use of pharmaceuticals, their presence in the aquatic environment, and the associated toxic effects, have raised concerns in recent years. In this work, a new multi-residue analytical method was developed and validated for the determination of 10 pharmaceuticals in wastewaters using online solid-phase extraction (online SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The compounds included in the method were antineoplastics (cabazitaxel, docetaxel, doxorubicin, etoposide, irinotecan, methotrexate, paclitaxel, and topotecan), renin inhibitors (aliskiren), and antidepressants (maprotiline). The method was developed through several experiments on four online SPE cartridges, three reversed phase chromatography columns, and four combinations of mobile phase components. Under optimal conditions, very low limits of detection (LODs) of 1.30 to 10.6 ng L⁻¹ were obtained. The method was repeatable, with relative standard deviations (RSD, %) for intraday and interday precisions ranged from 1.6 to 7.8 and from 3.3 to 13.2, respectively. Recovery values ranged from 78.4 to 111.4%, indicating the reproducibility of the method. Matrix effects were mainly presented as signal suppression, with topotecan and doxorubicin being the two most affected compounds (31.0% signal suppression). The proposed method was successfully applied to hospital effluents, detecting methotrexate (4.7–9.3 ng L⁻¹) and maprotiline (11.2–23.1 ng L⁻¹). Due to the shorter overall run time of 15 min, including sample preparation, and reduced sample volume (0.9 mL), this on-line SPE-LC-MS/MS method was extremely convenient and efficient in comparison to the classical off-line SPE method. The proposed method was also highly sensitive and can be used for ultratrace quantification of the studied pharmaceuticals in wastewaters, providing useful data for effective environmental monitoring. Full article

Mobility of eukaryotic transposable elements (TEs) are finely regulated to avoid an excessive mutational load caused by their movement. The transposition of retrotransposons is usually regulated through the interaction of host- and TE-encoded proteins, with non-coding regions (LTR and 5′-UTR) of the transposon. Examples of new potent cis-acting sequences, identified and characterized in the non-coding regions of retrotransposons, include the insulator of gypsy and Idefix, and the enhancer of ZAM of Drosophila melanogaster. Recently we have shown that in the 5′-UTR of the LTR-retrotransposon ZAM there is a sequence structured in tandem-repeat capable of operating as an insulator both in Drosophila (S2R⁺) and human cells (HEK293). Here, we test the hypothesis that tandem repeated 5′-UTR of a different LTR-retrotransposon could accommodate similar regulatory elements. The comparison of the 5′-UTR of some LTR-transposons allowed us to identify a shared motif of 13 bp, called Transposable Element Redundant Motif (TERM). Surprisingly, we demonstrated, by Yeast One-Hybrid assay, that TERM interacts with the D. melanogaster ribosomal protein RpL22. The Drosophila RpL22 has additional Ala-, Lys- and Pro-rich sequences at the amino terminus, which resembles the carboxy-terminal portion of histone H1 and histone H5. For this reason, it has been hypothesized that RpL22 might have two functions, namely the role in organizing the ribosome, and a potential regulatory role involving DNA-binding similar to histone H1, which represses transcription in Drosophila. In this paper, we show, by two independent sets of experiments, that DmRpL22 is able to directly and specifically bind DNA of Drosophila melanogaster. Full article