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Apnea diving is characterized by extreme hypoxia and hypercapnia. Possible pathophysiological processes concerning the cardiovascular system are not yet fully understood. Hypoxia has effects on triglyceride metabolism and circulating blood lipids. To date, in voluntary apnea divers, no short-time hypoxia expositions focusing on plasma triglycerides, lipoprotein and cholesterol derived oxysterols levels have been performed. We hypothesize that short time hypoxemia leads to altered triglyceride, cholesterol, and oxysterol plasma levels in voluntary apnea divers.

Reliability is of serious concern for high-k dielectrics used in advanced memory applications. In this study, the time dependent dielectric breakdown behavior is investigated for metal-insulator-metal capacitors with amorphous ZrAlxOy thin films as insulator and TiN as electrodes. Constant voltage stress measurements over seven orders of magnitude in time show that the power-law model is appropriate for lifetime extrapolation. The voltage acceleration parameter increases with decreasing temperature, and the thermal activation energy, Ea, increases with decreasing stress voltage, both translating to a gain in lifetime under product operation conditions. [ABSTRACT FROM AUTHOR]

The time-dependent dielectric breakdown behavior of metal-insulator-metal capacitors with amorphous Zr[Al.sub.x][O.sub.y] thin films is discussed. The high-k dielectric is shown to be widely used in the dynamic random access memory metal-insulator-metal capacitor and other applications.

Abstract: A broad compositional range of the dielectric material Zr1− x Hf x O2 was evaluated with respect to its applicability in DRAM storage capacitors. The paper reports on phase composition, crystallization behavior, and electrical properties of the mixed system in planar metal-insulator-metal (MIM) capacitors. Admixture of HfO2 into ZrO2 proved to stabilize the deposition process at high temperatures without degrading the dielectric properties of the film. Compared to pure ZrO2 the 30–40% HfO2 containing films showed improved scalability (capacitance equivalent thickness 0.73nm at 8*10−9 A/cm2) as well as improved reliability. [Copyright &y& Elsevier]

Staphylococcus aureus CC239-MRSA-III is an ancient pandemic strain of hospital-associated, methicillin-resistant S. aureus that spread globally for decades and that still can be found in some parts of the world. In Kuwait, microarray-based surveillance identified from 2019 to 2022 a series of isolates of a hitherto unknown variant of this strain that carried a second set of recombinase genes, ccrA/B-2. To elucidate the structure of its SCCmec element, two isolates were subjected to nanopore sequencing. This revealed, in addition to ccrA/B-2, several SCC-associated genes including speG (spermidine N acetyltransferase) and a gene encoding a large "E-domain containing protein" (dubbed as edcP-SCC). This gene contained three regions consisting of multiple repeating units. In terms of sequence and structure it was similar but not identical to the biofilm-related aap gene from S. epidermidis. A review of published sequences identified edcP-SCC in eighteen genome sequences of S. aureus, S. epidermidis and S. capitis, and frequently it appears in a similar cluster of genes as in the strains sequenced herein. Isolates also carried a prophage with the adhesion factor sasX/sesI and aminoglycoside resistance genes. This is consistent with an affiliation to the "South-East Asian" Clade of CC239. The emergence of edcP-SCC and sasX-positive CC239 strain shows that, against a global trend towards community-associated MRSA, the ancient pandemic CC239 hospital strain still continues to evolve and to cause outbreaks., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Staphylococcus argenteus is a recently described staphylococcal species that is related to Staphylococcus aureus but lacks the staphyloxanthin operon. It is able to acquire both resistance markers such as the SCC mec elements and mobile genetic elements carrying virulence-associated genes from S. aureus . This includes those encoding the Panton-Valentine leukocidin (PVL), which is associated mainly with severe and/or recurrent staphylococcal skin and soft tissue infections. Here, we describe the genome sequences of two PVL-positive, mecA -negative S. argenteus sequence type (ST) 2250 isolates from the United Arab Emirates in detail. The isolates were found in a dental clinic in the United Arab Emirates (UAE). Both were sequenced using Oxford Nanopore Technology (ONT). This demonstrated the presence of temperate bacteriophages in the staphylococcal genomes, including a PVL prophage. It was essentially identical to the published sequence of phiSa2wa_st78 (GenBank NC_055048), a PVL phage from an Australian S. aureus clonal complex (CC) 88 isolate. Besides the PVL prophage, one isolate carried another prophage and the second isolate carried two additional prophages, whereby the region between these two prophages was inverted. This "flipped" region comprised about 1,083,000 bp, or more than a third of the strain's genome, and it included the PVL prophage. Prophages were induced by Mitomycin C treatment and subjected to transmission electron microscopy (TEM). This yielded, in accordance to the sequencing results, one or, respectively, two distinct populations of icosahedral phages. It also showed prolate phages which presumptively might be identified as the PVL phage. This observation highlights the significance bacteriophages have as agents of horizontal gene transfer as well as the need for monitoring emerging staphylococcal strains, especially in cosmopolitan settings such as the UAE.

One of the greatest challenges to the use of molecular methods for diagnostic purposes is the detection of target DNA that is present only in low concentrations. One major factor that negatively impacts accuracy, diagnostic sensitivity, and specificity is the sample matrix, which hinders the attainment of the required detection limit due to the presence of residual background DNA. To address this issue, various methods have been developed to enhance sensitivity through targeted pre-amplification of marker sequences. Diagnostic sensitivity to the single molecular level is critical, particularly when identifying bloodstream infections. In cases of clinically manifest sepsis, the concentration of bacteria in the blood may reach as low as one bacterial cell/CFU per mL of blood. Therefore, it is crucial to achieve the highest level of sensitivity for accurate detection. In the present study, we have established a method that fills the analytical gap between low concentrations of molecular markers and the minimum requirements for molecular testing. For this purpose, a sample preparation of whole blood samples with a directly downstream pre-amplification was developed, which amplifies specific species and resistance markers in a multiplex procedure. When applying pre-amplification techniques, the sensitivity of the pathogen detection in whole blood samples was up to 100 times higher than in non-pre-amplified samples. The method was tested with blood samples that were spiked with several Gram-positive and Gram-negative bacterial pathogens. By applying this method to artificial spiked blood samples, it was possible to demonstrate a sensitivity of 1 colony-forming unit (CFU) per millilitre of blood for S. aureus and E. faecium . A detection limit of 28 and 383 CFU per ml of blood was achieved for E. coli and K. pneumoniae , respectively. If the sensitivity is also confirmed for real clinical blood samples from septic patients, the novel technique can be used for pathogen detection without cultivation, which might help to accelerate diagnostics and, thus, to decrease sepsis mortality rates.

Objective: Animal studies suggest that prebiotic, plant-derived nutrients could improve homoeostatic and hedonic brain functions through improvements in microbiome-gut-brain communication. However, little is known if these results are applicable to humans. Therefore, we tested the effects of high-dosed prebiotic fibre on reward-related food decision-making in a randomised controlled within-subject cross-over study and assayed potential microbial and metabolic markers., Design: 59 overweight young adults (19 females, 18-42 years, body mass index 25-30 kg/m 2 ) underwent functional task MRI before and after 14 days of supplementary intake of 30 g/day of inulin (prebiotics) and equicaloric placebo, respectively. Short chain fatty acids (SCFA), gastrointestinal hormones, glucose/lipid and inflammatory markers were assayed in fasting blood. Gut microbiota and SCFA were measured in stool., Results: Compared with placebo, participants showed decreased brain activation towards high-caloric wanted food stimuli in the ventral tegmental area and right orbitofrontal cortex after prebiotics (preregistered, family wise error-corrected p <0.05). While fasting blood levels remained largely unchanged, 16S-rRNA sequencing showed significant shifts in the microbiome towards increased occurrence of, among others, SCFA-producing Bifidobacteriaceae , and changes in >60 predicted functional signalling pathways after prebiotic intake. Changes in brain activation correlated with changes in Actinobacteria microbial abundance and associated activity previously linked with SCFA production, such as ABC transporter metabolism., Conclusions: In this proof-of-concept study, a prebiotic intervention attenuated reward-related brain activation during food decision-making, paralleled by shifts in gut microbiota., Trial Registration Number: NCT03829189., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Staphylococcus aureus is a versatile pathogen that does not only occur in humans but also in various wild and domestic animals, including several avian species. When characterizing S. aureus isolates from waterfowl, isolates were identified as atypical CC133 by DNA microarray analysis. They differed from previously sequenced CC133 strains in the presence of the collagen adhesin gene cna ; some also showed a different capsule type and a deviant spa type. Thus, they were subjected to whole-genome sequencing. This revealed multiple insertions of large regions of DNA from other S. aureus lineages into a CC133-derived backbone genome. Three distinct strains were identified based on the size and extent of these inserts. One strain comprised two small inserts of foreign DNA up- and downstream of oriC ; one of about 7000 nt or 0.25% originated from CC692 and the other, at ca. 38,000 nt or 1.3% slightly larger one was of CC522 provenance. The second strain carried a larger CC692 insert (nearly 257,000 nt or 10% of the strain's genome), and its CC522-derived insert was also larger, at about 53,500 nt or 2% of the genome). The third strain carried an identical CC692-derived region (in which the same mutations were observed as in the second strain), but it had a considerably larger CC522-like insertion of about 167,000 nt or 5.9% of the genome. Both isolates of the first, and two out of four isolates of the second strain also harbored a hemolysin-beta-integrating prophage carrying "bird-specific" virulence factors, ornithine cyclodeaminase D0K6J8 and a putative protease D0K6J9. Furthermore, isolates had two different variants of SCC elements that lacked mecA/mecC genes. These findings highlight the role of horizontal gene transfer in the evolution of S. aureus facilitated by SCC elements, by phages, and by a yet undescribed mechanism for large-scale exchange of core genomic DNA.

The aim of this study was to comprehensively characterise S. aureus from the Caribbean Islands of Trinidad and Tobago, and Jamaica. A total of 101 S. aureus / argenteus isolates were collected in 2020, mainly from patients with skin and soft tissue infections. They were characterised by DNA microarray allowing the detection of ca. 170 target genes and assignment to clonal complexes (CC)s and strains. In addition, the in vitro production of Panton-Valentine leukocidin (PVL) was examined by an experimental lateral flow assay. Two isolates were identified as S. argenteus , CC2596. The remaining S. aureus isolates were assigned to 21 CCs. The PVL rate among methicillin-susceptible S. aureus (MSSA) isolates was high (38/101), and 37 of the 38 genotypically positive isolates also yielded positive lateral flow results. The isolate that did not produce PVL was genome-sequenced, and it was shown to have a frameshift mutation in agrC . The high rate of PVL genes can be attributed to the presence of a known local CC8-MSSA clone in Trinidad and Tobago (n = 12) and to CC152-MSSA (n = 15). In contrast to earlier surveys, the USA300 clone was not found, although one MSSA isolate carried the ACME element, probably being a mecA -deficient derivative of this strain. Ten isolates, all from Trinidad and Tobago, were identified as MRSA. The pandemic ST239-MRSA-III strain was still common (n = 7), but five isolates showed a composite SCC mec element not observed elsewhere. Three isolates were sequenced. That showed a group of genes (among others, speG , crzC, and ccrA / B -4) to be linked to its SCC element, as previously found in some CC5- and CC8-MRSA, as well as in S. epidermidis . The other three MRSA belonged to CC22, CC72, and CC88, indicating epidemiological connections to Africa and the Middle East.

Staphylococcus ( S. ) aureus colonizes up to 30% of all humans and can occasionally cause serious infections. It is not restricted to humans as it can also often be found in livestock and wildlife. Recent studies have shown that wildlife strains of S. aureus usually belong to other clonal complexes than human strains and that they might differ significantly with regard to the prevalence of genes encoding antimicrobial resistance properties and virulence factors. Here, we describe a strain of S. aureus isolated from a European badger ( Meles meles ). For molecular characterisation, DNA microarray-based technology was combined with various next-generation sequencing (NGS) methods. Bacteriophages from this isolate were induced with Mitomycin C and characterized in detail by transmission electron microscopy (TEM) and NGS. The S. aureus isolate belonged to ST425 and had a novel spa repeat sequence (t20845). It did not carry any resistance genes. The uncommon enterotoxin gene see was detected in one of its three temperate bacteriophages. It was possible to demonstrate the induction of all three prophages, although only one of them was expected to be capable of excision based on its carriage of the excisionase gene xis . All three bacteriophages belonged to the family Siphoviridae . Minor differences in size and shape of their heads were noted in TEM images. The results highlight the ability of S. aureus to colonize or infect different host species successfully, which can be attributed to a variety of virulence factors on mobile genetic elements, such as bacteriophages. As shown in the strain described herein, temperate bacteriophages not only contribute to the fitness of their staphylococcal host by transferring virulence factors, but also increase mobility among themselves by sharing genes for excision and mobilization with other prophages.

Background: It is suspected that microbiome-derived trimethylamine N-oxide (TMAO) may enhance platelet responsiveness and accordingly be thrombophilic. The purpose of this prospective observational study is to evaluate TMAO in patients with subarachnoid hemorrhage (SAH) and compare it with a control group. A secondary aim was to investigate TMAO in the cerebrospinal fluid (CSF) from SAH patients. This should provide a better understanding of the role of TMAO in the pathogenesis of SAH and its thrombotic complications., Methods: The study included patients with diagnosed spontaneous SAH recruited after initial treatment on admission and patients with nerve, nerve root, or plexus disorders serving as controls. Blood samples were gathered from all patients at recruitment. Additionally, sampling of SAH patients in the intensive care unit continued daily for 14 days. The CSF was collected out of existing external ventricular drains whenever possible., Results: Thirty-four patients diagnosed with SAH, and 108 control patients participated in this study. Plasma TMAO levels at baseline were significantly lower in the SAH group (1.7 μmol/L) compared to the control group (2.9 μmol/L). TMAO was detectable in the CSF (0.4 μmol/L) and significantly lower than in plasma samples of the SAH group at baseline. Plasma and CSF TMAO levels correlated positively. The TMAO levels did not differ significantly during the observation period of 15 days., Conclusions: Although we assumed that patients with higher TMAO levels were at higher risk for SAH a priori, plasma TMAO levels were lower in patients with SAH compared with control subjects with nerve, nerve root, or plexus disorders on admission to the hospital. A characteristic pattern of plasma TMAO levels in patients with SAH was not found., (© 2023. The Author(s).)

Background: Non-cholesterol sterols, as well as plant sterols, cross the blood-brain barrier and, thus, can be incorporated into cell membranes, affecting the cell's inflammatory response. The aim of our work was to develop an analytical protocol for a quantitative assessment of the sterol composition within the membrane microdomains of microglia., Methods: A protocol for cell membrane isolation using OptiPrep TM gradient ultracentrifugation, in combination with a targeted mass spectrometry (LC-MS/MS)-based assay, was developed and validated for the quantitative analysis of free sterols in microglia cell membranes., Results: Utilizing an established LC-MS/MS assay, cholesterol and seven non-cholesterol sterols were analyzed with a limit of detection from 0.001 to 0.05 mg/L. Applying the detergent-free isolation of SIM-A9 microglia cell membranes, cholesterol (CH), desmosterol (DE), lanosterol (LA) stigmasterol (ST), beta-sitosterol (SI) and campesterol (CA) were quantified with coefficients of variations between 6 and 29% (fractions 4-6, n = 5). The highest concentrations of non-CH sterols within the microglia plasma membranes were found in the microdomain region (DE>LA>SI>ST>CA), with ratios to CH ranging from 2.3 to 435 lower abundancies., Conclusion: By applying our newly developed and validated analytical protocol, we show that the non-CH sterol concentration is about 38% of the total sterol content in microglia membrane microdomains. Further investigations must clarify how changes in the non-sterol composition influence membrane fluidity and cell signaling.

The present study aims to characterise clinical MRSA isolates from a tertiary care centre in Egypt's second-largest city, Alexandria. Thirty isolates collected in 2020 were genotypically characterised by microarray to detect their resistance and virulence genes and assign them to clonal complexes (CC) and strains. Isolates belonged to 11 different CCs and 14 different strains. CC15-MRSA-[V+ fus ] (n = 6), CC1-MRSA-[V+ fus+tir+ccrA/B-1 ] (PVL+) (n = 5) as well as CC1-MRSA-[V+ fus+tir+ccrA/B-1 ] and CC1153-MRSA-[V+ fus ] (PVL+) (both with n = 3) were the most common strains. Most isolates (83%) harboured variant or composite SCC mec V or VI elements that included the fusidic acid resistance gene fusC . The SCC mec [V+ fus+tir+ccrA/B -1] element of one of the CC1 isolates was sequenced, revealing a presence not only of fusC but also of blaZ , aacA-aphD and other resistance genes. PVL genes were also common (40%). The hospital-acquired MRSA CC239-III strain was only found twice. A comparison to data from a study on strains collected in 2015 (Montelongo et al., 2022) showed an increase in fusC and PVL carriage and a decreasing prevalence of the CC239 strain. These observations indicate a diffusion of community-acquired strains into hospital settings. The beta-lactam use in hospitals and the widespread fusidic acid consumption in the community might pose a selective pressure that favours MRSA strains with composite SCC mec elements comprising mecA and fusC . This is an unsettling trend, but more MRSA typing data from Egypt are required.

Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species ( Macaca mulatta , M. assamensis , and M. sylvanus ) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2 / etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton-Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.

Microbiome-derived trimethylamine N -oxide (TMAO) has been associated with platelet hyperreactivity and subsequent atherogenesis. Whether physiological TMAO-levels influence platelet-derived lipid mediators remains unknown. Little is known about pre-analytic factors potentially influencing TMAO concentrations. We aimed at developing a quantitative LC-MS/MS method to investigate in-vivo and in-vitro pre-analytical factors in TMAO analysis to properly assess the proposed activating effect of TMAO on platelets. TMAO, betaine, carnitine, and choline were analyzed by HILIC-ESI-MS/MS within 6 min total run time. Method validation included investigation of reproducibility, recovery, sensitivity, and in-vitro pre-analytical factors. A 24-h monitoring experiment was performed, evaluating in-vivo pre-analytical factors like daytime or diet. Finally, the effects of different TMAO concentrations on platelet activation and corresponding alterations of platelet-derived eicosanoid release were analyzed. The method showed high reproducibility (CVs ≤ 5.3%), good recovery rates (96-98%), and negligible in-vitro pre-analytical effects. The influence of in-vivo pre-analytical factors on TMAO levels was not observable within the applied experimental conditions. We did not find any correlation between TMAO levels and platelet activation at physiological TMAO concentrations, whereas platelet-derived eicosanoids presented activation of the cyclooxygenase and lipoxygenase pathways. In contrast to previously published results, we did not find any indications regarding diet dependency or circadian rhythmicity of TMAO levels. Our results do not support the hypothesis that TMAO increases platelet responsiveness via the release of lipid-mediators.

Apolipoprotein E (apoE) occurs on the majority of plasma lipoproteins and plays a major role in the lipid metabolism in the periphery and in the central nervous system. ApoE is a polymorphic protein with three common isoforms, apoE2, apoE3 and apoE4, derived from respective alleles ε2, ε3 and ε4. The aim of this study was to develop a sample pretreatment protocol combined with rapid mass spectrometry (MS)-based assay for simultaneous apolipoprotein profiling and apoE phenotype identification. This assay was validated in 481 samples from patients with stable atherosclerotic cardiovascular disease (ASCVD) and applied to study association with mild cognitive impairment (MCI) in the LIFE Adult study, including overall 690 study subjects. Simultaneous quantification of 8−12 major apolipoproteins including apoA-I, apoB-100 and apoE could be performed within 6.5 min. Phenotyping determined with the developed MS assay had good agreement with the genotyping by real-time fluorescence PCR (97.5%). ApoE2 isoform was associated with the highest total apoE concentration compared to apoE3 and apoE4 (p < 0.001). In the subgroup of diabetic atherosclerotic cardiovascular disease (ASCVD) patients, apoE2 isoform was related to higher apoC-I levels (apoE2 vs. apoE3, p < 0.05), while in the subgroup of ASCVD patients under statin therapy apoE2 was related to lower apoB-100 levels (apoE2 vs. apoE3/apoE4, p < 0.05). A significant difference in apoE concentration observed between mild cognitive impairment (MCI) subjects and controls was confirmed for each apoE phenotype. In conclusion, this study provides evidence for the successful implementation of an MS-based apoE phenotyping assay, which can be used to assess phenotype effects on plasma lipid and apolipoprotein levels.

The phylogenetic tree of the Staphylococcus aureus complex consists of several distinct clades and the majority of human and veterinary S. aureus isolates form one large clade. In addition, two divergent clades have recently been described as separate species. One was named Staphylococcus argenteus , due to the lack of the "golden" pigment staphyloxanthin. The second one is S. schweitzeri , found in humans and animals from Central and West Africa. In late 2021, two additional species, S. roterodami and S. singaporensis , have been described from clinical samples from Southeast Asia. In the present study, isolates and their genome sequences from wild Straw-coloured fruit bats ( Eidolon helvum ) and a Diamond firetail ( Stagonopleura guttata , an estrildid finch) kept in a German aviary are described. The isolates possessed staphyloxanthin genes and were closer related to S. argenteus and S. schweitzeri than to S. aureus . Phylogenetic analysis revealed that they were nearly identical to both, S. roterodami and S. singaporensis . We propose considering the study isolates, the recently described S. roterodami and S. singaporensis as well as some Chinese strains with MLST profiles stored in the PubMLST database as different clonal complexes within one new species. According to the principle of priority we propose it should be named S. roterodami . This species is more widespread than previously believed, being observed in West Africa, Southeast Asia and Southern China. It has a zoonotic connection to bats and has been shown to be capable of causing skin and soft tissue infections in humans. It is positive for staphyloxanthin, and it could be mis-identified as S. aureus (or S. argenteus ) using routine procedures. However, it can be identified based on distinct MLST alleles, and " S. aureus " sequence types ST2470, ST3135, ST3952, ST3960, ST3961, ST3963, ST3965, ST3980, ST4014, ST4075, ST4076, ST4185, ST4326, ST4569, ST6105, ST6106, ST6107, ST6108, ST6109, ST6999 and ST7342 belong to this species., Competing Interests: DG is employed by a company, Illumina, but he performed experiments for this study before commencing this employment. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Monecke, Schaumburg, Shittu, Schwarz, Mühldorfer, Brandt, Braun, Collatz, Diezel, Gawlik, Hanke, Hotzel, Müller, Reinicke, Feßler and Ehricht.)

Lipids are increasingly recognized as bioactive mediators of extracellular vesicle (EV) functions. However, while EV proteins and nucleic acids are well described, EV lipids are insufficiently understood due to lack of adequate quantitative methods. We adapted an established targeted and quantitative mass spectrometry (LC-MS/MS) method originally developed for analysis of 94 eicosanoids and seven polyunsaturated fatty acids (PUFA) in human plasma. Additionally, the influence of freeze-thaw (FT) cycles, injection volume, and extraction solvent were investigated. The modified protocol was applied to lipidomic analysis of differently polarized macrophage-derived EVs. We successfully quantified three PUFAs and eight eicosanoids within EVs. Lipid extraction showed reproducible PUFA and eicosanoid patterns. We found a particularly high impact of FT cycles on EV lipid profiles, with significant reductions of up to 70%. Thus, repeated FT will markedly influence analytical results and may alter EV functions, emphasizing the importance of a standardized sample pretreatment protocol for the analysis of bioactive lipids in EVs. EV lipid profiles differed largely depending on the polarization of the originating macrophages. Particularly, we observed major changes in the arachidonic acid pathway. We emphasize the importance of a standardized sample pretreatment protocol for the analysis of bioactive lipids in EVs.

Staphylococcus aureus can be a harmless coloniser, but it can also cause severe infections in humans, livestock and wildlife. Regarding the latter, only few studies have been performed and knowledge on virulence factors is insufficient. The aim of the present study was to study S. aureus isolates from deceased wild beavers (Castor fiber). Seventeen isolates from eleven beavers, found in Germany and Austria, were investigated. Antimicrobial and biocide susceptibility tests were performed. Isolates were characterised using S. aureus-specific DNA microarrays, spa typing and whole-genome sequencing. From two isolates, prophages were induced by mitomycin C and studied by transmission electron microscopy. Four isolates belonged to clonal complex (CC) 8, CC12, and CC398. Twelve isolates belonged to CC1956 and one isolate was CC49. The CC49 and CC1956 isolates carried distinct lukF/S genes related to the Panton-Valentine leukocidin (PVL) from human isolates of S. aureus. These genes were located on related, but not identical, Siphovirus prophages. The beavers, from which those isolates originated, suffered from abscesses, purulent organ lesions and necrotising pneumonia, i.e., clinical manifestations resembling symptoms of severe PVL-associated disease in humans. It might thus be assumed that the "Beaver Leukocidin (BVL, lukF/S-BV)"-positive strains are beaver-specific pathogens, and further studies on their clinical role as well as on a possible transmissibility to other species, including humans, are warranted., (© 2021. The Author(s).)

Plant sterols (PSs) cannot be synthesized in mammals and are exclusively diet-derived. PSs cross the blood-brain barrier and may have anti-neuroinflammatory effects. Obesity is linked to lower intestinal uptake and blood levels of PSs, but its effects in terms of neuroinflammation-if any-remain unknown. We investigated the effect of high-fat diet-induced obesity on PSs in the brain and the effects of the PSs campesterol and β-sitosterol on in vitro microglia activation. Sterols (cholesterol, precursors, PSs) and polyunsaturated fatty acid-derived lipid mediators were measured in the food, blood, liver and brain of C57BL/6J mice. Under a PSs-poor high-fat diet, PSs levels decreased in the blood, liver and brain (>50%). This effect was reversible after 2 weeks upon changing back to a chow diet. Inflammatory thromboxane B2 and prostaglandin D2 were inversely correlated to campesterol and β-sitosterol levels in all brain regions. PSs content was determined post mortem in human cortex samples as well. In vitro, PSs accumulate in lipid rafts isolated from SIM-A9 microglia cell membranes. In summary, PSs levels in the blood, liver and brain were associated directly with PSs food content and inversely with BMI. PSs dampen pro-inflammatory lipid mediators in the brain. The identification of PSs in the human cortex in comparable concentration ranges implies the relevance of our findings for humans.

Obesity arising from excessive dietary fat intake is a risk factor for cognitive decline, dementia and neurodegenerative diseases, including Alzheimer's disease. Here, we studied the effect of long-term high-fat diet (HFD) (24 weeks) and return to normal diet (ND) on behavioral features, microglia and neurons in adult male C57BL/6J mice. Consequences of HFD-induced obesity and dietary changes on general health (coat appearance, presence of vibrissae), sensory and motor reflexes, learning and memory were assessed by applying a phenotypic assessment protocol, the Y maze and Morris Water Maze test. Neurons and microglia were histologically analyzed within the mediobasal hypothalamus, hippocampus and frontal motor cortex after long-term HFD and change of diet. Long periods of HFD caused general health issues (coat alterations, loss of vibrissae), but did not affect sensory and motor reflexes, emotional state, memory and learning. Long-term HFD increased the microglial response (increased Iba1 fluorescence intensity, percentage of Iba1-stained area and Iba1 gene expression) within the hypothalamus, but not in the cortex and hippocampus. In neither of these regions, neurodegeneration or intracellular lipid droplet accumulation was observed. The former alterations were reversible in mice whose diet was changed from HFD to ND. Taken together, long periods of excessive dietary fat alone do not cause learning deficits or spatial memory impairment, though HFD-induced obesity may have detrimental consequences for cognitive flexibility. Our data confirm the selective responsiveness of hypothalamic microglia to HFD., Competing Interests: The authors have declared that no competing interests exist.

The prevalence of multidrug resistant, extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is increasing worldwide. The present study aimed to provide an overview of the multidrug resistance phenotype and genotype of ESBL-producing Escherichia coli ( E. coli ) isolates of livestock and wild bird origin in Greece. Nineteen phenotypically confirmed ESBL-producing E. coli strains isolated from fecal samples of cattle ( n = 7), pigs ( n = 11) and a Eurasian magpie that presented resistance to at least one class of non β-lactam antibiotics, were selected and genotypically characterized. A DNA-microarray based assay was used, which allows the detection of various genes associated with antimicrobial resistance. All isolates harbored bla CTX-M-1/15 , while bla TEM was co-detected in 13 of them. The AmpC gene bla MIR was additionally detected in one strain. Resistance genes were also reported for aminoglycosides in all 19 isolates, for quinolones in 6, for sulfonamides in 17, for trimethoprim in 14, and for macrolides in 8. The intI1 and/or tnpISE cp1 genes, associated with mobile genetic elements, were identified in all but two isolates. This report describes the first detection of multidrug resistance genes among ESBL-producing E. coli strains retrieved from feces of cattle, pigs, and a wild bird in Greece, underlining their dissemination in diverse ecosystems and emphasizing the need for a One-Health approach when addressing the issue of antimicrobial resistance.

Polyunsaturated fatty acids (PUFAs) and eicosanoids are important mediators of inflammation. The functional role of eicosanoids in metabolic-syndrome-related diseases has been extensively studied. However, their role in neuroinflammation and the development of neurodegenerative diseases is still unclear. The aim of this study was the development of a sample pretreatment protocol for the simultaneous analysis of PUFAs and eicosanoids in mouse liver and brain. Liver and brain samples of male wild-type C57BL/6J mice (11-122 mg) were used to investigate conditions for tissue rinsing, homogenization, extraction, and storage. A targeted liquid chromatography-negative electrospray ionization tandem mass spectrometry method was applied to quantify 7 PUFAs and 94 eicosanoids. The final pretreatment protocol consisted of a 5-min homogenization step by sonication in 650 μL n-hexane/2-propanol (60:40 v/v) containing 2,6-di-tert-butyl-4-methylphenol at 50 μg/mL. Homogenates representing 1 mg tissue were extracted in a single step with n-hexane/2-propanol (60:40 v/v) containing 0.1% formic acid. Autoxidation was prevented by addition of 2,6-di-tert-butyl-4-methylphenol at 50 μg/mL and keeping the samples at 4 °C during sample preparation. Extracts were dried under nitrogen and reconstituted in liquid chromatography eluent before analysis. Recovery was determined to range from 45% to 149% for both liver and brain tissue. Within-run and between-run variability ranged between 7% and 18% for PUFAs and between 1% and 24% for eicosanoids. In liver, 7 PUFAs and 15 eicosanoids were quantified; in brain, 6 PUFAs and 21 eicosanoids had significant differences within the brain substructures. In conclusion, a robust and reproducible sample preparation protocol for the multiplexed analysis of PUFAs and eicosanoids by liquid chromatography-tandem mass spectrometry in liver and discrete brain substructures was developed.

Background: The popularity of apneic diving is continually growing. As apnea diving substantially burdens the cardiovascular system, special focus is warranted. Regarding inflammation processes and associated inflammatory-related diseases (e.g., cardiovascular diseases), eicosanoids play an important role. This study aims to investigate polyunsaturated fatty acids (PUFAs) and eicosanoids in voluntary apnea divers, and so to further improve understanding of pathophysiological processes focusing on proinflammatory effects of temporarily hypercapnic hypoxia.., Methods: The concentration of PUFAs and eicosanoids were investigated in EDTA plasma in apnea divers (n=10) before and immediately after apnea, 0.5 hour and four hours later, applying liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Results: Mean age was 41±10 years, and divers performed a mean breath-hold time of 317±111 seconds. PUFAs, eicosanoids and related lipids could be classified in four different kinetical reaction groups following apnea. The first group (e.g., Ω-6 and Ω-3-PUFAs) showed an immediate concentration increase followed by a decrease below baseline four hours after apnea. The second group (e.g., thromboxane B2) showed a slower increase, with its maximum concentration 0.5 hour post-apnea followed by a decrease four hours post-apnea. Group 3 (9- and 13-hydroxyoctadecadienoic acid) is characterized by two concentration increase peaks directly after apnea and four hours afterward compared to baseline. Group 4 (e.g., prostaglandin D2) shows no clear response., Conclusion: Changes in the PUFA metabolism after even a single apnea revealed different kinetics of pro- and anti-inflammatory regulations and changes for oxidative stress levels. Due to the importance of these mediators, apnea diving should be evaluated carefully and be performed only with great caution against the background of cardiovascular diseases and inflammation processes., Competing Interests: The authors of this paper declare no conflicts of interest exist with this submission., (Copyright© Undersea and Hyperbaric Medical Society.)

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI(+)-MS/MS) assay was developed and qualified for analyzing 35 analytes of the cholesterol metabolism, including free cholesterol, 17 free, non-esterified oxysterols and 17 free and conjugated bile acids in plasma and cerebrospinal fluid. As internal standards, 25 commercially available stable deuterium-labeled analogs of the analytes were used. Pre-analytical investigations included stability tests of analyte concentrations affected by different anticoagulation additives: lithium heparin-, citrate-, EDTA-K 3 -stabilized plasma and serum, and the stability in EDTA whole blood at RT. This LC-ESI(+)-MS/MS method was successfully applied for the analysis of paired serum/cerebrospinal fluid samples of patients with and without blood-brain barrier disturbance, as well as of 100 plasma samples of a LIFE-Adult study sub-cohort. A fast and simple sample preparation including protein precipitation and on-line solid-phase extraction was developed. As little as 55 μL of human plasma/serum or cerebrospinal fluid were needed for the analysis. It was possible to separate isomeric oxysterols and bile acids within 23 min using a C18 core-shell column. The assay is capable of quantifying in a linear range of 0.8-250 ng mL -1 for free hydroxycholesterols, 0.2-10 ng mL -1 for dihydroxycholesterols, 0.2-500 ng mL -1 for bile acids and 16-2000 μg mL -1 for cholesterol with acceptable accuracy and precision. In cerebrospinal fluid one free oxysterols, five free and five conjugated bile acids could be quantified. No significant differences between patients with and without blood-brain barrier disturbance were obtained. In the LIFE-Adult sub-cohort two free oxysterols, four free and seven conjugated bile acids could be quantified in EDTA plasma. Men showed significantly higher concentrations of 26-OHC than women (p = 0.035). Furthermore, in women lower levels of cholic acid, glycocholic acid, glycodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid and higher levels of taurocholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid/hyodeoxycholic acid were quantified., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Laborious sample pretreatment of biological samples represents the most limiting factor for the translation of targeted proteomics assays from research to clinical routine. An optimized method for the simultaneous quantitation of 12 major apolipoproteins (apos) combining on-line SPE and fast LC-MS/MS analysis in 6.5 min total run time was developed, reducing the manual sample pretreatment time of 3 μL serum or plasma by 60%. Within-run and between-day imprecisions below 10 and 15% (n = 10) and high recovery rates (94-131%) were obtained applying the high-throughput setup. High-quality porcine trypsin was used, which outperformed cost-effective bovine trypsin regarding digestion efficiency. Comparisons with immunoassays and another LC-MS/MS assay demonstrated good correlation (Pearson's R: 0.81-0.98). Further, requirements on sample quality concerning sampling, processing, and long-term storage up to 1 year were investigated revealing significant influences of the applied sampling material and coagulant on quantitation results. Apo profiles of 1339 subjects of the LIFE-Adult-Study were associated with lifestyle and physiological parameters as well as establish parameters of lipid metabolism (e.g., triglycerides, cholesterol). Besides gender effects, most significant impact was seen regarding lipid-lowering medication. In conclusion, this novel highly standardized, high-throughput targeted proteomics assay utilizes a fast, simultaneous analysis of 12 apos from least sample amounts., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The increasing interest in the analysis of triglyceride (TG) species and the individual fatty acid (FA) composition requires expeditious and reliable quantification strategies. The utilization of flow injection analysis (FIA) coupled to quadrupole tandem mass spectrometry (MS/MS) for the simultaneous quantitation of TG and identification of FA composition facilitates the multiplexed verification of various biomarkers from small sample quantities. Enzymatic methods based on saponification and glycerol analysis are not suited for the determination of the FA distribution in TGs. This protocol proposes a procedure for the establishment of a relative quantitation method for middle- to high-abundance plasma TGs and the corresponding FA composition. Essential topics as FIA-MS/MS method development as well as sample preparation and validation strategies are described in detail.