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  • 1
    In: Nature Communications, Springer Science and Business Media LLC, Vol. 14, No. 1 ( 2023-12-02)
    Abstract: Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.
    Type of Medium: Online Resource
    ISSN: 2041-1723
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2023
    detail.hit.zdb_id: 2553671-0
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  • 2
    In: Droplet, Wiley, Vol. 3, No. 3 ( 2024-07)
    Abstract: Varying the chemical consistency of acoustically levitated droplets opens up an in situ study of chemical and biochemical reactions in small volumes. However, the optimization of the mixing time and the minimization of the positional instability induced by solution dispensing are necessary for practical applications such as the study of the transient state of macromolecules crystallography during the ligand binding processes. For this purpose, we study the inertial mixing in a configuration compatible with the room‐temperature crystallography using the acoustic levitation diffractometer, therein solution drops ejected at high velocity collide and coalesce with droplets dispensed on acoustically levitated and rotating polymer thin‐film sample holders. With the proposed method, we are able to achieve the mixing time of ∼0.1 s for sub‐micro and a few microliter droplets. The observed short mixing time is ascribed to the rapid penetration of the solution into the droplets and confirmed by a computational fluid dynamic simulation. The demonstrated accelerated solution mixing is tested in a pilot time‐lapse protein crystallography experiment using the acoustic levitation diffractometer. The results indicate the detection of transient ligand binding state within 2 s after the solution dispensing, suggesting the feasibility of the proposed method for studying slow biochemical processes.
    Type of Medium: Online Resource
    ISSN: 2731-4375 , 2731-4375
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2024
    detail.hit.zdb_id: 3172821-2
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  • 3
    In: Scientific Reports, Springer Science and Business Media LLC, Vol. 12, No. 1 ( 2022-04-25)
    Type of Medium: Online Resource
    ISSN: 2045-2322
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2022
    detail.hit.zdb_id: 2615211-3
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  • 4
    In: Scientific Reports, Springer Science and Business Media LLC, Vol. 12, No. 1 ( 2022-03-30)
    Abstract: Acoustic levitation has attracted attention in terms of chemical and biochemical analysis in combination with various analytical methods because of its unique container-less environment for samples that is not reliant on specific material characteristics. However, loading samples with very high viscosity is difficult. To expand the scope, we propose the use of polymer thin films as sample holders, whereby the sample is dispensed on a film that is subsequently loaded onto an acoustic levitator. When applied for protein crystallography experiments, rotation controllability and positional stability are important prerequisites. We therefore study the acoustic levitation and rotation of thin films with an aspect ratio (the diameter-to-thickness ratio) of 80–240, which is an order of magnitude larger than those reported previously. For films with empirically optimized shapes, we find that it is possible to control the rotation speed in the range of 1–4 rotations per second while maintaining a positional stability of 12 ± 5 µm. The acoustic radiation force acting on the films is found to be a factor of 26–30 higher than that for same-volume water droplets. We propose use cases of the developed films for protein crystallography experiments and demonstrate data collections for large single crystal samples at room temperature.
    Type of Medium: Online Resource
    ISSN: 2045-2322
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2022
    detail.hit.zdb_id: 2615211-3
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  • 5
    In: IUCrJ, International Union of Crystallography (IUCr), Vol. 10, No. 6 ( 2023-11-01)
    Abstract: Fixed targets are a popular form of sample-delivery system used in serial crystallography at synchrotron and X-ray free-electron laser sources. They offer a wide range of sample-preparation options and are generally easy to use. The supports are typically made from silicon, quartz or polymer. Of these, currently, only silicon offers the ability to perform an aperture-aligned data collection where crystals are loaded into cavities in precise locations and sequentially rastered through, in step with the X-ray pulses. The polymer-based fixed targets have lacked the precision fabrication to enable this data-collection strategy and have been limited to directed-raster scans with crystals randomly distributed across the polymer surface. Here, the fabrication and first results from a new polymer-based fixed target, the micro-structured polymer fixed targets (MISP chips), are presented. MISP chips, like those made from silicon, have a precise array of cavities and fiducial markers. They consist of a structured polymer membrane and a stabilization frame. Crystals can be loaded into the cavities and the excess crystallization solution removed through apertures at their base. The fiducial markers allow for a rapid calculation of the aperture locations. The chips have a low X-ray background and, since they are optically transparent, also allow for an a priori analysis of crystal locations. This location mapping could, ultimately, optimize hit rates towards 100%. A black version of the MISP chip was produced to reduce light contamination for optical-pump/X-ray probe experiments. A study of the loading properties of the chips reveals that these types of fixed targets are best optimized for crystals of the order of 25 µm, but quality data can be collected from crystals as small as 5 µm. With the development of these chips, it has been proved that polymer-based fixed targets can be made with the precision required for aperture-alignment-based data-collection strategies. Further work can now be directed towards more cost-effective mass fabrication to make their use more sustainable for serial crystallography facilities and users.
    Type of Medium: Online Resource
    ISSN: 2052-2525
    Language: Unknown
    Publisher: International Union of Crystallography (IUCr)
    Publication Date: 2023
    detail.hit.zdb_id: 2754953-7
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  • 6
    In: European Heart Journal, Oxford University Press (OUP), ( 2024-05-20)
    Abstract: It has been reported that patients without standard modifiable cardiovascular (CV) risk factors (SMuRFs—diabetes, dyslipidaemia, hypertension, and smoking) presenting with first myocardial infarction (MI), especially women, have a higher in-hospital mortality than patients with risk factors, and possibly a lower long-term risk provided they survive the post-infarct period. This study aims to explore the long-term outcomes of SMuRF-less patients with stable coronary artery disease (CAD). Methods CLARIFY is an observational cohort of 32 703 outpatients with stable CAD enrolled between 2009 and 2010 in 45 countries. The baseline characteristics and clinical outcomes of patients with and without SMuRFs were compared. The primary outcome was a composite of 5-year CV death or non-fatal MI. Secondary outcomes were 5-year all-cause mortality and major adverse cardiovascular events (MACE—CV death, non-fatal MI, or non-fatal stroke). Results Among 22 132 patients with complete risk factor and outcome information, 977 (4.4%) were SMuRF-less. Age, sex, and time since CAD diagnosis were similar across groups. SMuRF-less patients had a lower 5-year rate of CV death or non-fatal MI (5.43% [95% CI 4.08–7.19] vs. 7.68% [95% CI 7.30–8.08] , P = 0.012), all-cause mortality, and MACE. Similar results were found after adjustments. Clinical event rates increased steadily with the number of SMuRFs. The benefit of SMuRF-less status was particularly pronounced in women. Conclusions SMuRF-less patients with stable CAD have a substantial but significantly lower 5-year rate of CV death or non-fatal MI than patients with risk factors. The risk of CV outcomes increases steadily with the number of risk factors.
    Type of Medium: Online Resource
    ISSN: 0195-668X , 1522-9645
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2024
    detail.hit.zdb_id: 2001908-7
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  • 7
    In: Rheumatology International, Springer Science and Business Media LLC, Vol. 42, No. 2 ( 2022-02), p. 261-271
    Abstract: The detection of antinuclear autoantibody (ANA) is dependent on many factors and varies between the populations. The aim of the study was first to assess the prevalence of ANA in the Polish adult population depending on age, sex and the cutoff threshold used for the results obtained. Second, we estimated the occurrence of individual types of ANA-staining patterns. We tested 1731 patient samples using commercially available IIFA using two cutoff thresholds of 1:100 and 1:160. We found ANA in 260 participants (15.0%), but the percentage of positive results strongly depended on the cutoff level. For a cutoff threshold 1:100, the positive population was 19.5% and for the 1:160 cutoff threshold, it was 11.7%. The most prevalent ANA-staining pattern was AC-2 Dense Fine speckled (50%), followed by AC-21 Reticular/AMA (14.38%) ANA more common in women (72%); 64% of ANA-positive patients were over 50 years of age. ANA prevalence in the Polish population is at a level observed in other highly developed countries and is more prevalent in women and elderly individuals. To reduce the number of positive results released, we suggest that Polish laboratories should set 1:160 as the cutoff threshold.
    Type of Medium: Online Resource
    ISSN: 0172-8172 , 1437-160X
    RVK:
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2022
    detail.hit.zdb_id: 1464208-6
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  • 8
    In: Biomarker Insights, SAGE Publications, Vol. 17 ( 2022-01), p. 117727192110667-
    Abstract: The anti-DFS70 autoantibodies are one of the most commonly and widely described agent of unknown clinical significance, frequently detected in healthy individuals. It is not known whether the DFS70 autoantibodies are protective or pathogenic. One of the factors suspected of inducing the formation of anti-DFS70 antibodies is increased oxidative stress. We evaluated the coexistence of anti-DFS70 antibodies with selected markers of oxidative stress and investigated whether these antibodies could be considered as indirect markers of oxidative stress. Methods: The intensity of oxidative stress was measured in all samples via indices of free-radical damage to lipids and proteins such as total oxidant status (TOS), concentrations of lipid hydroperoxides (LPH), lipofuscin (LPS), and malondialdehyde (MDA). The parameters of the non-enzymatic antioxidant system, such as total antioxidant status (TAS) and uric acid concentration (UA), were also measured, as well as the activity of superoxide dismutase (SOD). Based on TOS and TAS values, the oxidative stress index (OSI) was calculated. All samples were also tested with indirect immunofluorescence assay (IFA) and 357 samples were selected for direct monospecific anti DFS70 enzyme-linked immunosorbent assay (ELISA) testing. Results: The anti-DFS70 antibodies were confirmed by ELISA test in 21.29% of samples. Compared with anti-DFS70 negative samples we observed 23% lower concentration of LPH ( P = .038) and 11% lower concentration of UA ( P = .005). TOS was 20% lower ( P = .014). The activity of SOD was up to 5% higher ( P = .037). The Pearson correlation showed weak negative correlation for LPH, UA, and TOS and a weak positive correlation for SOD activity. Conclusion: In samples positive for the anti-DFS70 antibody a decreased level of oxidative stress was observed, especially in the case of samples with a high antibody titer. Anti-DFS70 antibodies can be considered as an indirect marker of reduced oxidative stress or a marker indicating the recent intensification of antioxidant processes.
    Type of Medium: Online Resource
    ISSN: 1177-2719 , 1177-2719
    Language: English
    Publisher: SAGE Publications
    Publication Date: 2022
    detail.hit.zdb_id: 2256754-9
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  • 9
    In: Praehistorische Zeitschrift, Walter de Gruyter GmbH, Vol. 89, No. 2 ( 2014-6-30), p. 261-279
    Abstract: Zusammenfassung: Forschungsgegenstand dieses Artikels ist ein Begräbnis- und Ritualplatz der Kugelamphoren-Kultur der Fundstätte 14 in Kowal (Zentral-Polen). Die Stätte umfasst einen Submegalithen sowie einen Ritualplatz mit Tiergräbern und einer menschlichen Bestattung. Der Komplex kann auf die Zeit zwischen 3250/3100 und 2400/2150 v. Chr. datiert werden. Analysen haben gezeigt, dass der Platz über eine lange Zeit benutzt wurde, und dass die durchgeführten Rituale sehr komplex waren. Beweise wurden gefunden, dass an einigen Stellen rituelle Handlungen ohne Unterbrechung über viele Jahre durchgeführt wurden und einzelne Tieropfer und Niederlegungen nur durch einen Zeitraum von wenigen Jahren voneinander getrennt waren. Die Fund-Struktur einiger Gruben zeigt eine sehr penible Organisation des geheiligten Platzes und ermöglicht an einigen Stellen sogar eine Unterscheidung in Nutz- und rituelle Zonen. Weitere wichtige Informationen wurden durch die Untersuchung einer menschlichen Grablege an dieser Stätte gewonnen. Die Analyse der Grabbeigaben hat deren Natur als rituelle Gaben gezeigt; weiter weist sie auf eine dominante Rolle von Tieren im täglichen Leben der verstorbenen Person hin. Wichtige Daten wurden durch die paläo-biologische Untersuchung der menschlichen Knochen gewonnen. Die paläo-serologische Analyse ergab die Blutgruppe der bestatteten Person. Dies ist der erste derartige Nachweis in der Geschichte der europäischen neolithischen Forschung. Bei den Ergebnissen der DNA-Analyse handelt es eines Vertreters der Kugelamphoren-Kultur in der Literatur und zudem um eine von nur wenigen solcher Untersuchungen eines neolithischen Menschen. Einige der nachgewiesenen Allele sind die ältesten Beispiele dieses speziellen Allel-Typs, die in menschlichen Überresten bis heute identifiziert wurden. Die DNA-Analysen belegen eine Laktose-Intoleranz der bestatteten Person, sie legen weiter eine genetische Verwandtschaft mit der Bevölkerung aus der Region des Fruchtbaren Halbmondes nahe. Die Analyse des stabilen Sauerstoff-Isotopen-Verhältnisses zeigt Muster für häufigeren Ortswechsel und das Entwöhnungsalter. Analysen von stabilen Kohlenstoff- und Stickstoff-Isotopen-Verhältnissen haben Rückschlüsse auf die Ernährung ermöglicht; besondere Aufmerksamkeit verdient die Tatsache, dass bis zu 25 % der Kohlenhydrat-haltigen Nahrung aus Hirse bestanden hat. Das ist der früheste Isotopen-gestützte Nachweis von Hirse in der menschlichen Ernährung in Mitteleuropa. Résumé: Le site 14 de Kowal en Pologne centrale, un complexe rituel appartenant à la culture des amphores globulaires, forme le sujet de cet article. Le site comporte un sub-mégalithe, une structure rituelle avec des dépôts d’animaux et une sépulture humaine. Il fut occupé pendant une longue période allant de 3250/3100 à 2400/2150 av. J.-C. et les rituels étaient complexes: certains furent célébrés au même endroit pendant longtemps, tandis que les sacrifices et dépôts d’animaux n’étaient séparés que par de courts intervalles de peutêtre quelques années. Le remplissage de plusieurs fosses démontre une organisation précise de l’espace sacré, et il a même été possible de faire une distinction entre les actes de déposition rituelle et les dépôts de type utilitaire à l’intérieur de ces fosses. L’étude de la sépulture humaine a également fourni d’importants indices. Il en ressort qu’un individu intentionnellement choisi parmi les membres de ‘l’aristocratie’ locale a été la victime d’un meurtre rituel. L’analyse du mobilier funéraire indique qu’il s’agit d’offrandes rituelles et suggère que les animaux jouaient un rôle déterminant dans la vie de cet individu. L’étude de ses ossements a fourni un important ensemble de données, y compris les résultats d’une analyse sérologique permettant d’identifier – pour la première fois pour le Néolithique européen – le groupe sanguin de cette homme. Dans une étude également pionnière pour la culture des amphores globulaires, l’analyse ADN a permis de décrire la composition génétique d’un de ses membres, une rareté dans les études sur le Néolithique. Certains allèles sont les plus anciens exemplaires d’un type particulier d’allèles jusqu’à présent découvert dans des restes humains. Les données de l’analyse de l’ADN de cet individu suggèrent qu’il était intolérant au lactose et qu’il avait des affinités génétiques avec le Croissant fertile. Les résultats de l’analyse des isotopes stables de l’oxygène peuvent être interprétés soit en termes de mobilité ou de sevrage. Ceux provenant de l’analyse des isotopes stables du carbone et de l’azote nous permettent de reconstruire l’alimentation de notre individu ; en particulier le millet fournissait jusqu’à 25 % de sa consommation en glucides. Ce résultat constitue le premier indice isotopique de la présence du millet dans l’alimentation des populations de l’Europe centrale. Abstract: Site 14 in Kowal (central Poland), a funerary and ritual place of the Globular amphora culture forms the subject of this article. The site includes a sub-megalith, a ritual feature with animal burials and a human burial. The complex can be dated to a period spanning from 3250/3100 to 2400/2150 BC; it was used over a long period, and the rituals carried out were complex. Rituals were performed over many years in certain areas, while individual acts of animal sacrifice and deposition may have been separated by a short interval of just a few years. The fill of some pits shows that the sacred space was carefully organised and it has been possible to distinguish between some utilitarian and ritual deposits within them. Other important information is provided by the study of a human burial discovered at the site. It is possible that a deliberately chosen individual from the tribal ‘aristocracy’ was the victim of a ritual murder. The analysis of the grave goods reveals that they were ritual offerings, and these suggest that animals played a dominant role in the deceased man’s daily life. The study of his bones provides important new insights, including those from a palaeo-serological analysis revealing his blood group, a first in the history of European Neolithic research. The results of DNA analysis constitute the first description of the genetic traits of a representative of the Globular amphora culture, among very few such DNA profiles available for a Neolithic person. Some of the alleles discovered are the oldest examples of particular alleles among those identified in human remains so far. DNA evidence suggests that the individual was lactose intolerant, and it demonstrates a genetic relationship with the Fertile Crescent. The analysis of stable oxygen isotope ratios shows patterns compatible with either residential mobility or weaning age. The analysis of stable carbon and nitrogen isotope ratios allowed us to reconstruct the individual’s diet; particular attention is drawn to the fact that up to 25 % of his carbohydrate diet consisted of millet. This is the earliest isotopic evidence for the presence of millet in the diet of people living in Central Europe.
    Type of Medium: Online Resource
    ISSN: 1613-0804 , 0079-4848
    RVK:
    Language: English
    Publisher: Walter de Gruyter GmbH
    Publication Date: 2014
    detail.hit.zdb_id: 2050013-0
    SSG: 6,11
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